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## **Meet the editor**

Ota Fuchs graduated from the Chemical Technological University, Prague, Czech Republic in 1971. He obtained his PhD in Biochemistry from Faculty of Natural Sciences, Charles University, Prague, in 1981. He is employed as a senior scientist at the Institute of Hematology and Blood Transfusion, Prague. He shortly worked as a visiting scientist in the Beatson Institute for Cancer

Research, Glasgow, UK, and in Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada. He was the principal investigator of five projects of Internal Grant Agency of Ministry of Health of Czech Republic and of one grant project of Grant Agency of Czech Republic. He is a member of Czech Society for Biochemistry and Molecular Biology and of Czech Medical Society J.E.Purkyne.

## Contents

#### **Preface XI**

	- Lale Olcay and Sevgi Yetgin

## Preface

Despite efforts in studies of the biology of myelodysplastic syndromes (MDS), a diagnosis of MDS in individual patient relies mainly on morphological and cytogenetic examinations of the bone marrow and blood. Genomic instability, gene mutations, deregulated gene expression, and epigenetic changes play the key role in the pathogenesis of MDS. High-throughput genetic tech‐ nologies such as single nucleotide polymorphism and comparative genomic hybridization and next-generation sequencing helped in performing mutation profiling but are preferentially used in clinical trials and not in routine laboratories. These laboratories are limited by the cost-effective care and interpretation barriers, and they do not currently use mutational profiling for diagnosis of MDS. Many mutations are not specific for MDS, and they also occur in other myeloid malig‐ nancies. Besides somatic mutations, cytokine aberrations, immune dysregulation, alterations in the bone marrow microenvironment (niche), abnormal RNA splicing, and changes in telomers and signal pathways are involved in the MDS pathogenesis.

This book provides a concise review of advances in myelodysplastic syndromes and will be use‐ ful not only to the researchers and clinicians involved in this topic but also to medical students. Introduction chapter is a brief review of current knowledge of MDS prognostication, pathogene‐ sis, and therapy. Chapter 2 discusses disorders mimicking MDS and difficulties in MDS diagno‐ sis. Chapter 3 analyzes the role of inflammation and immune suppression in the pathogenesis of MDS. The application of immunosuppressive treatment may relieve the autoimmune manifesta‐ tions but also improves hematopoiesis. Chapter 4 focuses on the 5q- syndrome first described by Van den Berghe et al. in 1974. Patients with 5q- syndrome have refractory anemia with del(5q) as the sole karyotypic abnormality and medullary blast count of less than 5 %. The del(5q) occurs in approximately 10–20 % of patients with de novo MDS. Pathogenesis of the 5q- syndrome has been intensively studied in last years, and immunomodulatory or cereblon-binding drug lenali‐ domide is used for the treatment of transfusion-dependent anemia in these lower-risk del(5q) MDS patients. MDS cases with del(5q) and additional karyotypic abnormalities and secondary MDS cases with del(5q) have a poor prognosis in contrast to the good prognosis of the 5q- syn‐ drome. Chapters 5 and 6 review higher-risk MDS, monosomy 7, and chronic myelomonocytic leu‐ kemia, treated by hypomethylating agents (azacitidine and decitabine). Allogeneic stem cell transplantation is the only treatment option with curative potential for higher-risk MDS patients but is limited by age of patients. Resistance to chemotherapy is a serious obstacle to the successful treatment. This important topic is discussed in an overview in Chapter 7.

Each chapter is written by scientists and clinicians with specific expertise in the field.

**Ota Fuchs, PhD** Institute of Hematology and Blood Transfusion Department of Genomics Prague, Czech Republic

## **Introductory Chapter: Myelodysplastic Syndromes**

#### Ota Fuchs

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64729

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral cytopenias, frequent karyotypic abnormalities, and risk of transformation to acute myeloid leukemia (AML) [1–5]. MDS are rare in young people, and median age of patients with MDS is approximately 70 years [6, 7]. Current management in MDS includes supportive care, drug therapy, and allogeneic stem cell transplantation. MDS patients older than age 70 years are not good candidates for allogeneic stem cell transplantation [8, 9]. The major complication of MDS patients after allogeneic stem cell transplantation with reduced intensity conditioning is relapse of malignant disease. Relapse can be predicted by monitoring of Wilms' tumor 1 (WT1) gene expression by real-time PCR and CD34+ donor chimerism analysis [10].

Single-nucleotide polymorphism array (SNP-A) analysis, standard metaphase cytogenetics, and rapid progress in flow cytometric analysis, genes mutation analysis, and gene expression profiling have identified key deregulated genes and signaling pathways important for accurate prognostication and risk stratification for individual patients with MDS [11–18]. The initial French–British–American (FAB) classification system of MDS was published in 1982 [19] and was later refined to the International Prognostic Scoring System (IPSS) [20] and to World Health Organization (WHO) Prognostic Scoring System (WPSS) [21, 22]. The new revised IPSS (IPSS-R) integrated marrow cytogenetic subset, marrow blast percentage, and depth of cytopenias (hemoglobin, platelet, and absolute neutrophil count) and was published in 2012 [23]. Validation of WPSS for MDS and comparison with IPSS-R has been recently described [24]. Two other prognostic systems for MDS subgroups (M.D. Anderson lower risk MDS prognostic scoring system; chronic myelomonocytic leukemia /CMML/ prognostic scoring system) exist [18, 25].

In lower risk MDS, treatment focuses on amelioration of consequences of cytopenias and transfusions and improving of quality of life. The first line of therapy of lower risk MDS with normal chromosome 5 is treatment with erythropoiesis-stimulating agents (erythropoietin)

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

with or without of granulocyte colony-stimulating factor [26]. Transfusion-dependent lower risk MDS patients with del(5q) are treated with immunomodulatory or cereblon-binding drug lenalidomide [27]. Thrombocytopenia occurring sometimes in combination with anemia or without anemia can be treated with romiplostin (thrombopoietin agonist) [27]. Neutropenia is treated with growth factors (G-CSF and GM-CSF) [27]. Higher-risk MDS if untreated have median survival only about 12 months. Two hypomethylating agents (azacitidine and decitabine) inhibit DNA methyltransferases 3A and 3B and reverse the aberrant methylation involved in MDS progression to AML. The development of novel therapeutic strategies in MDS is dependent on recent advances in the molecular pathogenesis of MDS [6, 16, 28–39]. Various combination therapies in MDS are also intensively studied [27, 40, 41].

#### **Author details**

#### Ota Fuchs\*

Address all correspondence to: Ota.Fuchs@uhkt.cz

Institute of Hematology and Blood Transfusion, Prague, Czech Republic

#### **References**


[8] Tamari R, Castro-Malaspina H. Transplant for MDS: challenges and emerging strat‐ egies. Best Pract Res Clin Haematol. 2015; 28: 43–54. doi:10.1016/j.beha.2014.11.006

with or without of granulocyte colony-stimulating factor [26]. Transfusion-dependent lower risk MDS patients with del(5q) are treated with immunomodulatory or cereblon-binding drug lenalidomide [27]. Thrombocytopenia occurring sometimes in combination with anemia or without anemia can be treated with romiplostin (thrombopoietin agonist) [27]. Neutropenia is treated with growth factors (G-CSF and GM-CSF) [27]. Higher-risk MDS if untreated have median survival only about 12 months. Two hypomethylating agents (azacitidine and decitabine) inhibit DNA methyltransferases 3A and 3B and reverse the aberrant methylation involved in MDS progression to AML. The development of novel therapeutic strategies in MDS is dependent on recent advances in the molecular pathogenesis of MDS [6, 16, 28–39].

Various combination therapies in MDS are also intensively studied [27, 40, 41].

Institute of Hematology and Blood Transfusion, Prague, Czech Republic

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[24] Della Porta MG, Tuechler H, Malcovati L, Schanz J, Sanz G, Garcia-Manero G, Solé F, Bennett JM, Bowen D, Fenaux P, Dreyfus F, Kantarjian H, Kuendgen A, Levis A, Cermak J, Fonatsch C, Le Beau MM, Slovak ML, Krieger O, Luebbert M, Maciejewski J, Magalhaes SM, Miyazaki Y, Pfeilstöcker M, Sekeres MA, Sperr WR, Stauder R, Tauro S, Valent P, Vallespi T, van de Loosdrecht AA, Germing U, Haase D, Greenberg PL, Cazzola M. Validation of WHO classification-based Prognostic Scoring System (WPSS) for myelodysplastic syndromes and comparison with the revised International Prognostic Scoring System (IPSS-R). A study of the International Working Group for Prognosis in Myelodysplasia (IWG-PM). Leukemia. 2015; 29: 1502–1513. doi:10.1038/

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## **Immune Dysregulation in Myelodysplastic Syndromes: Pathogenetic-Pathophysiologic Aspects and Clinical Consequences**

Argiris Symeonidis and Alexandra Kouraklis-Symeonidis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64618

#### **Abstract**

relapsed or refractory myeloid malignancies. Cancer. 2016; 122: 1871–1879. doi:10.1002/

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cncr.29986.

6 Myelodysplastic Syndromes

508. doi:10.1016/S1470-2045(16)00009-7.

e352. doi:10.14694/EDBK\_161221.

Des. 2016; 22: 2323–2332.

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders, in which the immune system plays a substantial pathogenetic role. Patients manifest frequent infections, mainly attributed to neutropenia, but sometimes opportunistic pathogens are isolated in non-neutropenic patients. They also exhibit autoimmune diseases or syndromes with a background of immune activation and various "abnormalities" of Tlymphocytes, B-lymphocytes, and NK cells. The most typical profile includes reduced total T lymphocytes (mainly CD4+ helper T-cells, resulting in decrease or inversion of the CD4/CD8 cell ratio) and impaired NK cell function. Many TH1 direction cytokines, and particularly sIL-2R, IL-6, and TNF-α are usually found increased in the serum and bone marrow, which have been strongly associated with advanced disease, anemia, and other disease-related features. Clonal origin of lymphocytes has been confirmed only in few cases. Mixed lymphocyte cultures and genomic assays have shown severely impaired immunoregulatory abnormalities, probably induced by the hematopoietic cells. In a minority of patients, immune activation is capable to prevent or delay clonal expan‐ sion, but these patients have more profound hematopoietic impairment. Immunosup‐ pressive treatment may not only relieve the autoimmune manifestations but also improve hematopoiesis. However, this kind of treatment is not well tolerated, is associated with severe infections, and in some cases may enhance AML evolution.

**Keywords:** myelodysplastic syndromes, pathogenesis, immune abnormalities, auto‐ immune diseases, immunosuppressive treatment

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

Myelodysplastic syndromes (MDS) are diseases emerging from somatic mutations of a pluripotent hematopoietic stem cell, affecting its functional capacity for maturation and differentiation, but preserving it alive and capable to escape from apoptotic signals. The affected cell creates a clone, gradually suppressing nonclonal cells, and finally dominating in the bone marrow. Clonal cells are prone to additional genetic events, promoting survival advantage and further impairing differentiation and maturation, thus generating a neoplastic phenotype, with the evolution to acute myelogenous leukemia (AML). Since such genetic events occur serially, but with a variable evolutionary potential, MDS represent probably the best *in vivo* model of "step-by-step" progression from a premalignant state to a high-potential neoplasm, such as AML. The multiparametric study of MDS can draw messages and conclusions, potentially applicable for the pathogenesis and pathophysiology of all types of neoplasia.

One rather unexpected aspect of MDS pathophysiology is the strong involvement of the immune system. Soon after the initial description of MDS, many "immune abnormalities" were reported in the literature. The "easy" initial interpretation of the participation of the immune system to the dysplastic clone was quickly changed, in favor of other explanations. But even now, a clear interpretation and a definite treatment strategy for these "abnormalities" have not been established. In this chapter, we describe the spectrum of "*immune abnormalities*" of MDS and briefly discuss treatment approaches, targeting the immune system. Besides a thorough literature review, we have used our personal experience, based on the study of more than 1500 patients. Thus, this chapter includes also original data, presented at various meetings, but not yet published as full papers, emerged from personal/institutional research activities at the Department of Hematology of the University Hospital of Patras.

#### **2. Infections unrelated to the severity of neutropenia among patients with MDS**

Infections of various etiologies are common among MDS patients and represent one of the major presenting features, but also a leading cause of morbidity and mortality. Beside the usually advanced patient age and comorbidity, the major predisposing factor in many retrospective studies analyzing the frequency and severity of infections is the depth of neutropenia. Functional neutrophil abnormalities have also been reported, such as impaired locomotion and chemotaxis, reduced complement receptor-1 and -3 expressions, and reduced enzymatic armamentarium, resulting in impaired respiratory burst and reduced bactericidal and fungicidal capacity. These defects have been observed in all types of MDS but are more frequent in the higher risk groups [1]. However, severe common and opportunistic infections may be manifested in nonneutropenic patients. In these cases, besides the functional neutrophil impairment, various acquired defects of the adaptive immunity, affecting immunocompetent cell populations have been proposed as predisposing factors. Finally, transfusions, transfu‐ sion-induced iron overload, and the newer treatment modalities, such as lenalidomide and hypomethylating agents, may hamper immune functions and contribute to the development of infections.

There are several published cases or small series of patients manifesting pyogenic collections/ abscesses, not only at common sites (perianal, splenic, liver), but also at rare and uncommon (pararenal, intramuscular, paracolic, etc.), without the development of strong inflammatory reaction [2, 3]. It has been suggested that mature granulocytes of MDS patients may not produce effective inflammatory reaction to eliminate pathogens, and induce the formation of granulomas or abscesses [1, 2]. MDS patients, even when nonneutropenic, may exhibit delayed healing of infections and have increased intracellular neutrophil collagenase activity, irre‐ spective of WHO or IPSS subgroup [3].

Other patients manifest bacterial, viral, and fungal infections, from rare/opportunistic pathogens, before any immunosuppressive or cytotoxic treatment, similar to those encoun‐ tered among patients with underlying congenital or acquired immunodeficiency. Among such rare bacterial pathogens, coagulase-negative *Staphylococci*, rare Enterococci, *Myc. aviumintracellulare*, *Myc. Kansasii*, *Myc. Malmoense*, *Bacillus cereus*, *Corynebacterium* and *Phenylobacte‐ rium* spp., *Aeromonas hydrophila*, *Brevundimonas diminuta*, *Rhodococcus corynebacterioides*, and *Bordetella hinzii* are included. Among viral, fungal, and other pathogens, CMV and EBV reactivation, HHV-6 infection, JCV-induced progressive multifocal leukoencephalopathy (PML), *Pneumocystis iirovecii*, and *Legionella pneumophila* are included. Invasive fungal infec‐ tions are rather uncommon and may emerge during the myelosuppression, which follows cytotoxic chemotherapy and/or allogeneic stem cell transplantation, in patients receiving prophylactically or therapeutically strong combinations of antibacterial antibiotics. However, many cases have been reported in the absence of these recognized predisposing factors. Involved pathogens are both, yeasts, including *C. albicans*, *Candida* non-*albicans* spp., and *Cryptococcus neoformans* and molds, such as *Aspergillus* and zygomycetes, and the usually affected organs are the lungs, liver, spleen, and central nervous system (CNS), and more rarely the skin, soft tissues, and other organs.

#### **3. Association of autoimmune and immune-mediated diseases, with the manifestation of MDS**

#### **3.1. Clinical syndromes of immune overactivity associated with myelodysplastic syndromes**

#### *3.1.1. Clearly autoimmune diseases*

**1. Introduction**

8 Myelodysplastic Syndromes

**MDS**

Myelodysplastic syndromes (MDS) are diseases emerging from somatic mutations of a pluripotent hematopoietic stem cell, affecting its functional capacity for maturation and differentiation, but preserving it alive and capable to escape from apoptotic signals. The affected cell creates a clone, gradually suppressing nonclonal cells, and finally dominating in the bone marrow. Clonal cells are prone to additional genetic events, promoting survival advantage and further impairing differentiation and maturation, thus generating a neoplastic phenotype, with the evolution to acute myelogenous leukemia (AML). Since such genetic events occur serially, but with a variable evolutionary potential, MDS represent probably the best *in vivo* model of "step-by-step" progression from a premalignant state to a high-potential neoplasm, such as AML. The multiparametric study of MDS can draw messages and conclusions, potentially

One rather unexpected aspect of MDS pathophysiology is the strong involvement of the immune system. Soon after the initial description of MDS, many "immune abnormalities" were reported in the literature. The "easy" initial interpretation of the participation of the immune system to the dysplastic clone was quickly changed, in favor of other explanations. But even now, a clear interpretation and a definite treatment strategy for these "abnormalities" have not been established. In this chapter, we describe the spectrum of "*immune abnormalities*" of MDS and briefly discuss treatment approaches, targeting the immune system. Besides a thorough literature review, we have used our personal experience, based on the study of more than 1500 patients. Thus, this chapter includes also original data, presented at various meetings, but not yet published as full papers, emerged from personal/institutional research

**2. Infections unrelated to the severity of neutropenia among patients with**

Infections of various etiologies are common among MDS patients and represent one of the major presenting features, but also a leading cause of morbidity and mortality. Beside the usually advanced patient age and comorbidity, the major predisposing factor in many retrospective studies analyzing the frequency and severity of infections is the depth of neutropenia. Functional neutrophil abnormalities have also been reported, such as impaired locomotion and chemotaxis, reduced complement receptor-1 and -3 expressions, and reduced enzymatic armamentarium, resulting in impaired respiratory burst and reduced bactericidal and fungicidal capacity. These defects have been observed in all types of MDS but are more frequent in the higher risk groups [1]. However, severe common and opportunistic infections may be manifested in nonneutropenic patients. In these cases, besides the functional neutrophil impairment, various acquired defects of the adaptive immunity, affecting immunocompetent cell populations have been proposed as predisposing factors. Finally, transfusions, transfu‐ sion-induced iron overload, and the newer treatment modalities, such as lenalidomide and

applicable for the pathogenesis and pathophysiology of all types of neoplasia.

activities at the Department of Hematology of the University Hospital of Patras.

Only few years after the recognition of MDS as separate entities and the proposal of their first classification system (FAB classification), it was obvious that they were associated with increased frequency of various immune abnormalities, either abnormal laboratory findings, such as organ- and non-organ-specific autoantibodies, or true clinical syndromes or diseases, reflecting severely impaired adaptive immunity.

Among the autoimmune or immune-mediated clinical syndromes, described in association with MDS, Coombs-positive immunohemolytic anemia (AIHA) [4], immune thrombocytope‐ nia (ITP) [5], Evans' syndrome, autoimmune neutropenia, and pure red cell aplasia (usually drug-related) are included. AIHA is somewhat more common, and is usually of warm type, associated with mild to moderate chronic hemolysis [4]. It can be automatically presented or triggered by red blood cell transfusions. Treatment is mainly based on corticosteroids, but response rate is lower than in the idiopathic cases, and even when a "complete" response is achieved, this cannot be objectively evaluated due to coexistence of the basic disease, which has as a major manifestation anemia, although not clearly hemolytic. When corticosteroids are ineffective or associated with unacceptable toxicity, cyclosporine-A, mofetil mycophenolate, or pulses of vinca alkaloids may be used. In some instances immunosuppressive treatment may be accompanied by trilineage response, improving also neutrophil and platelet counts.

Immune thrombocytopenia particularly of chronic type, when manifested in elderly patients may mimic true MDS, particularly when there is additional underlying comorbidity and patients also have anemia of chronic disease. In such instances, the differential diagnosis is difficult and this is clearly an overlapping area of hematological disorders [6]. More compli‐ cating is the fact that these two different entities may share some common pathogenetic features, concerning premature megakaryocyte cell death [7]. However, true immune throm‐ bocytopenia with high titers of antiplatelet autoantibodies may be the presenting feature [6, 8] or may complicate the course of a previously diagnosed MDS [9]. In some instances, ITP may precede and MDS may follow some months or years, even after the achievement of complete response of the ITP. Thrombocytopenia has been related to higher amount of glycocalicin and platelet-associated IgG, higher MPV, more advanced disease, and worse prognosis in patients with MDS. The occurrence of ITP has been more frequently reported in chronic myelomonocytic leukemia (CMML) and the del-5q syndrome, whereas relatively severe thrombocytopenia, with mild-moderate or absence of anemia and neutropenia has been reported among patients with isolated del-20q [10]. Retrospective evaluation of 123 patients with CMML revealed the presence of auto-/hyperimmune disorders in 19.5% of them, compared to 3–4% incidence in the general population [11]. CMML has been considered the MDS, most frequently associated with "paraneoplastic" manifestations. Finally, high frequen‐ cy of hypocomplementemia, often associated with severe cytopenia, particularly in patients with lower risk MDS has been reported, suggesting the possible contribution of autoimmune mechanisms in its pathogenesis [12].

#### *3.1.2. Common clinical syndromes with a dominantly immune pathogenesis*

Dominant position among the immune hyperactivity/autoimmunity syndromes possess the various systemic vasculitides, such as febrile neutrophilic dermatosis (Sweet's syndrome) [13], other leucocytoclastic vasculitides, and necrotizing panniculitis, most commonly localized in the skin and accompanied by rashes or resulting in extended skin ulcerations. Large vessel arteritis (Takayasu's disease), aortitis, and other organ-specific vasculitides, such as Wegener's granulomatosis, have been reported. Additional cutaneous manifestations associated with the occult or prominent presence of an MDS include granulomatous eruptions, pyoderma gangrenosum, erythema nodosum, erythema elevatum diutinum, bullous pemphigoid, cutaneous lupus, Behçet's disease, dermatomyositis, and Raynaud's syndrome.

nia (ITP) [5], Evans' syndrome, autoimmune neutropenia, and pure red cell aplasia (usually drug-related) are included. AIHA is somewhat more common, and is usually of warm type, associated with mild to moderate chronic hemolysis [4]. It can be automatically presented or triggered by red blood cell transfusions. Treatment is mainly based on corticosteroids, but response rate is lower than in the idiopathic cases, and even when a "complete" response is achieved, this cannot be objectively evaluated due to coexistence of the basic disease, which has as a major manifestation anemia, although not clearly hemolytic. When corticosteroids are ineffective or associated with unacceptable toxicity, cyclosporine-A, mofetil mycophenolate, or pulses of vinca alkaloids may be used. In some instances immunosuppressive treatment may be accompanied by trilineage response, improving also neutrophil and platelet counts.

Immune thrombocytopenia particularly of chronic type, when manifested in elderly patients may mimic true MDS, particularly when there is additional underlying comorbidity and patients also have anemia of chronic disease. In such instances, the differential diagnosis is difficult and this is clearly an overlapping area of hematological disorders [6]. More compli‐ cating is the fact that these two different entities may share some common pathogenetic features, concerning premature megakaryocyte cell death [7]. However, true immune throm‐ bocytopenia with high titers of antiplatelet autoantibodies may be the presenting feature [6, 8] or may complicate the course of a previously diagnosed MDS [9]. In some instances, ITP may precede and MDS may follow some months or years, even after the achievement of complete response of the ITP. Thrombocytopenia has been related to higher amount of glycocalicin and platelet-associated IgG, higher MPV, more advanced disease, and worse prognosis in patients with MDS. The occurrence of ITP has been more frequently reported in chronic myelomonocytic leukemia (CMML) and the del-5q syndrome, whereas relatively severe thrombocytopenia, with mild-moderate or absence of anemia and neutropenia has been reported among patients with isolated del-20q [10]. Retrospective evaluation of 123 patients with CMML revealed the presence of auto-/hyperimmune disorders in 19.5% of them, compared to 3–4% incidence in the general population [11]. CMML has been considered the MDS, most frequently associated with "paraneoplastic" manifestations. Finally, high frequen‐ cy of hypocomplementemia, often associated with severe cytopenia, particularly in patients with lower risk MDS has been reported, suggesting the possible contribution of autoimmune

mechanisms in its pathogenesis [12].

10 Myelodysplastic Syndromes

*3.1.2. Common clinical syndromes with a dominantly immune pathogenesis*

cutaneous lupus, Behçet's disease, dermatomyositis, and Raynaud's syndrome.

Dominant position among the immune hyperactivity/autoimmunity syndromes possess the various systemic vasculitides, such as febrile neutrophilic dermatosis (Sweet's syndrome) [13], other leucocytoclastic vasculitides, and necrotizing panniculitis, most commonly localized in the skin and accompanied by rashes or resulting in extended skin ulcerations. Large vessel arteritis (Takayasu's disease), aortitis, and other organ-specific vasculitides, such as Wegener's granulomatosis, have been reported. Additional cutaneous manifestations associated with the occult or prominent presence of an MDS include granulomatous eruptions, pyoderma gangrenosum, erythema nodosum, erythema elevatum diutinum, bullous pemphigoid,


**Table 1.** Clinical syndromes of auto-/hyperimmune basis associated with myelodysplasia.

Fever of unknown origin in the absence of any known underlying condition has been described in association with MDS and may be accompanied by mild lymphadenopathy and sarcoidictype noncaseating granulomata, high serum ferritin, and polyclonal hyper-γ-globulinemia. Various rheumatic manifestations may also be associated with an MDS and some patients are diagnosed following an initial presentation of a typical rheumatic disease. Remitting seronegative symmetrical synovitis with pitting edema has been reported as an initial presentation of MDS, with subsequent manifestation of relapsing polychondritis. Besides the more frequent than expected classical rheumatoid arthritis and systemic lupus erythematosus, seronegative migratory synovitis, various seropositive and seronegative polyarthritic syndromes, polymyositis, polymyalgia rheumatica, eosinophilic fasciitis, Sjögren's syndrome, and mixed connective tissue disease have also been reported.

Less common syndromes are noninfectious serosal effusions, usually pleural, but sometimes also pericarditis, chronic autoimmune hepatitis, Hashimoto's thyroiditis, Addison's disease, inflammatory bowel disease, glomerulonephritis and nephrotic syndrome, focal and segmen‐ tal glomerulosclerosis, chronic autoimmune pancreatitis, ulcerative colitis, and various syndromes reflecting immune-based inflammatory processes of the CNS, such as seizures, expressive aphasia and paresis, and peripheral demyelinating polyneuropathy [14]. Finally, noninfectious pulmonary infiltrates, sometimes typical for alveolar proteinosis and bronchio‐ litis obliterans organizing pneumonia (BOOP), in the absence of previous allogeneic trans‐ plantation have also been reported. **Table 1** summarizes the various auto-/hyperimmune syndromes, sometimes called "paraneoplastic," associated with MDS.

#### *3.1.3. Relapsing polychondritis*

Of particular interest is the syndrome of relapsing polychondritis, which, besides the presen‐ tation as an idiopathic autoimmune syndrome, has almost exclusively been reported in association with MDS and very rarely with other diseases. Thus, among newly diagnosed polychondritis, without any evidence for a hematological disorder, BM examination may reveal the presence of an as yet undiagnosed MDS [15]. Polychondritis is manifested as painful inflammation of the cartilaginous areas of the body, such as the external ear, the basal area of the nose and the nasal septum, the synovial cartilage, and the tracheal and bronchial cartila‐ ginous rings. Symptomatic period may persist for many days or some weeks, followed by resolution, but symptoms reappear after weeks or months. The cartilage is finally destroyed, resulting in anatomical malformation and functional disturbances. The syndrome may be associated with fever, renal, cardiovascular, or ocular manifestations, as well as by symptoms, related to organ-specific dysfunction, not clearly containing cartilaginous tissue [16]. Patho‐ genesis is clearly immune-based and may reflect immunological reaction against some marrow stromal elements, which also exist in the cartilaginous tissue. Prompt intervention with corticosteroids accelerates resolution of the inflammatory reaction and may reduce tissue destruction and malformations.

#### *3.1.4. Overview of patient series and epidemiological data on autoimmune manifestations*

Autoimmune diseases have been reported on average in 10–30% of MDS patients, in all age groups and disease subtypes sometimes more frequently among females and in patients with higher risk MDS or CMML [9, 17]. In some early studies, the frequency of true autoimmune diseases among series of MDS patients was not found increased compared to non-MDS subjects of similar age. Moreover, the detection of various autoantibodies, directed against erythrocytic, neutrophilic, and platelet components, in the serum of MDS patients has been disputed, whether it really represents an immune abnormality, and has been attributed to the advanced patient age to thorough searching processes or to alloimmunization from the frequent transfusions. It has been suggested, although not proved, that similar results could be obtained from multiply transfused non-MDS patients of advanced age [18]. In another study, patients exhibiting immune abnormalities were younger and had mainly therapyrelated MDS with complex chromosomal aberrations [9]. Among the reported cases with available cytogenetics, trisomy 8 cases are rather overrepresented, but in the majority of described series of patients no clear preponderance of any demographic, cytogenetic, or histological feature was found [19]. In some studies, the frequency of autoimmune diseases is higher, but there is the possibility of misinterpretation of dysplastic bone marrow changes attributed to the advanced patient age or to the underlying autoimmune disease as indicative of primary MDS [17]. In a large French multicenter retrospective analysis of 123 MDS patients, exhibiting systemic inflammatory and autoimmune diseases (SIADs), vasculitic syndromes were more frequently encountered among CMML, and a comparison of this group with 665 patients without such manifestations revealed that patients exhibiting SIADs were younger, male, without RARS, with higher risk disease, and a poor karyotype, but without survival difference [20].

Various rheumatic manifestations may also be associated with an MDS and some patients are diagnosed following an initial presentation of a typical rheumatic disease. Remitting seronegative symmetrical synovitis with pitting edema has been reported as an initial presentation of MDS, with subsequent manifestation of relapsing polychondritis. Besides the more frequent than expected classical rheumatoid arthritis and systemic lupus erythematosus, seronegative migratory synovitis, various seropositive and seronegative polyarthritic syndromes, polymyositis, polymyalgia rheumatica, eosinophilic fasciitis, Sjögren's syndrome,

Less common syndromes are noninfectious serosal effusions, usually pleural, but sometimes also pericarditis, chronic autoimmune hepatitis, Hashimoto's thyroiditis, Addison's disease, inflammatory bowel disease, glomerulonephritis and nephrotic syndrome, focal and segmen‐ tal glomerulosclerosis, chronic autoimmune pancreatitis, ulcerative colitis, and various syndromes reflecting immune-based inflammatory processes of the CNS, such as seizures, expressive aphasia and paresis, and peripheral demyelinating polyneuropathy [14]. Finally, noninfectious pulmonary infiltrates, sometimes typical for alveolar proteinosis and bronchio‐ litis obliterans organizing pneumonia (BOOP), in the absence of previous allogeneic trans‐ plantation have also been reported. **Table 1** summarizes the various auto-/hyperimmune

Of particular interest is the syndrome of relapsing polychondritis, which, besides the presen‐ tation as an idiopathic autoimmune syndrome, has almost exclusively been reported in association with MDS and very rarely with other diseases. Thus, among newly diagnosed polychondritis, without any evidence for a hematological disorder, BM examination may reveal the presence of an as yet undiagnosed MDS [15]. Polychondritis is manifested as painful inflammation of the cartilaginous areas of the body, such as the external ear, the basal area of the nose and the nasal septum, the synovial cartilage, and the tracheal and bronchial cartila‐ ginous rings. Symptomatic period may persist for many days or some weeks, followed by resolution, but symptoms reappear after weeks or months. The cartilage is finally destroyed, resulting in anatomical malformation and functional disturbances. The syndrome may be associated with fever, renal, cardiovascular, or ocular manifestations, as well as by symptoms, related to organ-specific dysfunction, not clearly containing cartilaginous tissue [16]. Patho‐ genesis is clearly immune-based and may reflect immunological reaction against some marrow stromal elements, which also exist in the cartilaginous tissue. Prompt intervention with corticosteroids accelerates resolution of the inflammatory reaction and may reduce tissue

*3.1.4. Overview of patient series and epidemiological data on autoimmune manifestations*

Autoimmune diseases have been reported on average in 10–30% of MDS patients, in all age groups and disease subtypes sometimes more frequently among females and in patients with higher risk MDS or CMML [9, 17]. In some early studies, the frequency of true autoimmune diseases among series of MDS patients was not found increased compared to non-MDS

and mixed connective tissue disease have also been reported.

syndromes, sometimes called "paraneoplastic," associated with MDS.

*3.1.3. Relapsing polychondritis*

12 Myelodysplastic Syndromes

destruction and malformations.

Autoimmune manifestations may not be a single clinical syndrome, but a clustering of two or more autoimmune or immune-based conditions may occur in the same patient. Autoantibod‐ ies most frequently found are either organ-specific or non-organ-specific, such as rheumatoid factor, antinuclear antibodies, antineutrophil cytoplasmic (cANCA), or antineutrophil perinuclear antibodies (pANCA), the last two been associated with various vasculitides. Interferon regulatory factor-1 (IRF-1) mRNA expression was found 10-folds increased in MDS patients with autoimmune manifestations, in sharp difference to those without, and to normal controls. It was therefore suggested that absence of IRF-1 expression may be a protective mechanism preventing autoimmunity in MDS [21]. However, from the prognostic point of view although patients with autoimmune manifestations have similar overall survival compared to patients without those with higher IRF-1 expression have longer survival [22].

There is no agreement whether autoimmune manifestations influence prognosis. This is because the severity of autoimmune diseases and conditions, supervened by a MDS, may be substantially diverse and prevent their evaluation as an additional prognostic factor. The majority of retrospective studies tend to demonstrate a survival advantage for patients not exhibiting (auto)immune abnormalities as compared to those who did [23]; however, other studies did not show any difference [9]. In a Japanese study, patients with immune abnor‐ malities had more frequent infections, faster leukemic transformation, and shorter survival. Autoimmune diseases usually respond partially or temporarily to immunosuppressive treatment, but they may relapse and follow the basic disease activity [18]. In other instances they may persist throughout the course of the MDS and demand unaffordable corticosteroid doses to be controlled. In any case, autoimmune diseases remit permanently with allogeneic stem cell transplantation. Remission of autoimmune manifestations has been associated with improved survival, although in some studies it may accelerate evolution to AML. Furthermore, achievement of remission and return to the MDS stage may induce relapse of the autoimmune condition and in general the manifestation of autoimmunity follows the dysplastic phase and disappear during evolution to AML [24].

In the largest retrospective epidemiologic and prognostic analysis of about 1400 patients, 28% exhibited hyper/autoimmune manifestations and the most prominent was hypothyroidism associated with Hashimoto's thyroiditis (12% of the total population or 44% of the autoimmune syndromes), followed by immune thrombocytopenia (12%), rheumatoid arthritis (10%), and psoriasis (7%). Autoimmune conditions were more frequent among females with lower risk disease, and less transfusion dependent. The probability for AML transformation was lower and the median survival significantly higher for patients with autoimmune diseases (60 versus 45 months), and in multivariate analysis, adjusted for age and IPSS, the manifestation of an autoimmune syndrome was an independent favorable prognostic factor [23].

#### **4. Numerical abnormalities of lymphocytes in patients with MDS**

#### **4.1. T-lymphocyte abnormalities**

Many investigational studies have focused on various parameters of adaptive immunity in MDS. In the majority of patients, peripheral blood lymphopenia, mainly CD4+ cell lympho‐ penia, and to a lesser degree or at all, CD8+ cell reduction, frequently resulting in reduction or inversion of the CD4/CD8 cell ratio has been recognized. These findings have not been associated with specific FAB subtypes or any other clinical or laboratory feature [10, 20]. Many studies have confirmed the previous findings, particularly in patients with RAEB, and demonstrated severe functional T-cell impairment in terms of sluggish reaction to mitogenic stimuli and increased radiosensitivity, reflecting impaired DNA repair, implying that these defects might impact on patient hematopoiesis [25].

An initial approach for the interpretation of the numerical imbalance of T-cell subsets was that they might be attributed to multiple red blood cell transfusions, since in some early studies a correlation of the severity of T-lymphocyte abnormalities with transfusion intensity was reported [26]. However, it soon became clear that T-lymphocyte imbalance was present already at baseline, before any medical intervention, and therefore this finding might most probably be a disease feature. T cells of MDS patients synthesize lower amounts of the TH1 direction cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ), following mitogenic stimulation, respond inadequately to IL-2 and cooperate inefficiently with B lymphocytes in the induction of immunoglobulin production [24–28]. Studies of NK cell function have always reported reduced cytotoxic and cytolytic activity against cellular targets, as well as impaired both complement-dependent (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) [27]; however, in many other more recent studies, no abnormality in NK cells has been identified. We have investigated several immune function parameters at baseline on the same population of 81 patients with various MDS subtypes and we have shown that patients with RAEB had more profound CD3+ and CD4+ lymphopenia, and significantly lower and sometimes inverted CD4/CD8 cell ratio. We have also shown that CD3+ and CD8+ cell lymphopenia were associated with more frequent infections, higher AML evolution rate, and shorter overall survival [28]. A Japanese group confirmed decreased CD8+ cells in RA patients and inverted bone marrow CD4/CD8 cell ratio, with increased activated CD8+CD11α+ cells in all patients. RAEB patients had decreased marrow total T cells and all MDS patients had decreased marrow CD4+CD45RA+ naïve- and increased CD4+CD45RO+ memory T-helper cells, probably indicating impaired immune surveillance permitting the undisturbed evolution of the dysplastic clone. The prognostic significance of the numerical T-cell abnormalities on the above-mentioned issues was confirmed by other groups [19, 29]. Evaluation of lymphocyte subsets in bone marrow biopsies with specific immunostaining has not demonstrated any quantitative or qualitative T- and NK-cell abnormality, but only revealed increased B lym‐ phocytes in patients with higher risk disease and it has been suggested that identification of >3% B lymphocytes in the marrow biopsy is an adverse prognostic feature [30].

improved survival, although in some studies it may accelerate evolution to AML. Furthermore, achievement of remission and return to the MDS stage may induce relapse of the autoimmune condition and in general the manifestation of autoimmunity follows the dysplastic phase and

In the largest retrospective epidemiologic and prognostic analysis of about 1400 patients, 28% exhibited hyper/autoimmune manifestations and the most prominent was hypothyroidism associated with Hashimoto's thyroiditis (12% of the total population or 44% of the autoimmune syndromes), followed by immune thrombocytopenia (12%), rheumatoid arthritis (10%), and psoriasis (7%). Autoimmune conditions were more frequent among females with lower risk disease, and less transfusion dependent. The probability for AML transformation was lower and the median survival significantly higher for patients with autoimmune diseases (60 versus 45 months), and in multivariate analysis, adjusted for age and IPSS, the manifestation of an

autoimmune syndrome was an independent favorable prognostic factor [23].

**4. Numerical abnormalities of lymphocytes in patients with MDS**

Many investigational studies have focused on various parameters of adaptive immunity in MDS. In the majority of patients, peripheral blood lymphopenia, mainly CD4+ cell lympho‐ penia, and to a lesser degree or at all, CD8+ cell reduction, frequently resulting in reduction or inversion of the CD4/CD8 cell ratio has been recognized. These findings have not been associated with specific FAB subtypes or any other clinical or laboratory feature [10, 20]. Many studies have confirmed the previous findings, particularly in patients with RAEB, and demonstrated severe functional T-cell impairment in terms of sluggish reaction to mitogenic stimuli and increased radiosensitivity, reflecting impaired DNA repair, implying that these

An initial approach for the interpretation of the numerical imbalance of T-cell subsets was that they might be attributed to multiple red blood cell transfusions, since in some early studies a correlation of the severity of T-lymphocyte abnormalities with transfusion intensity was reported [26]. However, it soon became clear that T-lymphocyte imbalance was present already at baseline, before any medical intervention, and therefore this finding might most probably be a disease feature. T cells of MDS patients synthesize lower amounts of the TH1 direction cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ), following mitogenic stimulation, respond inadequately to IL-2 and cooperate inefficiently with B lymphocytes in the induction of immunoglobulin production [24–28]. Studies of NK cell function have always reported reduced cytotoxic and cytolytic activity against cellular targets, as well as impaired both complement-dependent (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) [27]; however, in many other more recent studies, no abnormality in NK cells has been identified. We have investigated several immune function parameters at baseline on the same population of 81 patients with various MDS subtypes and we have shown that patients with RAEB had more profound CD3+ and CD4+ lymphopenia, and significantly lower and

disappear during evolution to AML [24].

14 Myelodysplastic Syndromes

**4.1. T-lymphocyte abnormalities**

defects might impact on patient hematopoiesis [25].


*Notes*: Results of the skin reaction to the Multitest CMI test, consisting of 7 common antigens (Tetanus, Diphtheria, Tuberculin, Candida, Trichophyton, Streptococcus, Proteus): patients with MDS as one group exhibited significantly reduced composite score, compared to healthy controls, matched for age and gender. Significant differences were found for all FAB MDS categories, with the exception of RA (unpublished data).Bold letters indicate statistically significant differences.

**Table 2.** Results of the skin reaction to Multitest CMI® in patients with MDS

Patients with MDS-RA and with aplastic anemia exhibit Th1 and Tc1 polarization of their immune activation [31]. Later this was confirmed also for patients with refractory cytopenia with multilineage dysplasia (RCMD) and was correlated with very high serum and marrow IFN-γ and tumor necrosis factor-α (TNF-α) levels, and high degree of apoptosis. Higher Th1/ Th2 and Tc1/Tc2 ratios have been observed in patients with lower risk IPSS and normal karyotype, but not in aneuploid karyotypes. Th1 polarization may not be a uniform finding in MDS patients, but concerns only a subgroup of RCMD with prominent CD8+ lymphopenia [32].

Activated T lymphocytes do not belong to the dysplastic/leukemic clone and express HLA-DR, CD25, CD45RO, and CD57, but not CD28 and CD62L. This antigenic profile is independent of disease subtype, prognostic classification, kind of cytogenetic abnormality, or any other feature [33]. Interestingly, both patients with lower risk MDS and those with aplastic anemia have increased T-lymphocyte counts and B lymphocytopenia compared to patients with highrisk MDS and to controls, but lower risk MDS patients exhibit stronger and uniform Th1/Tc1 polarization than those with aplastic anemia [34]. Moreover, bone marrow NK T-cell infiltrates express the activated effector T-cell phenotype CD8+CD57+CD28−CD62L− and the NK Clectin-family receptors NKG2D and CD244. These infiltrates represent oligoclonal expansions of autoreactive T cells, as this can be demonstrated by TCR clonality assays and are more prominently identified in the bone marrow than in the peripheral blood [35].

MDS patients also exhibit impairment of delayed cutaneous T-cell hypersensitivity, as this can be demonstrated with various skin patch tests, challenging reaction to common antigens. Most importantly, they may lose immunologic memory against potentially important antigens, such as tuberculin and *clostr*. *tetani* anatoxin, the clinical consequence of which is unclear [36]. **Table 2** resumes the results from our study of skin reactions to the multitest CMI® in 54 patients with MDS compared to 20 controls (unpublished data).

Further understanding of the immune dysregulation of MDS was achieved through investi‐ gation of the regulatory T cells (T-regs). T-regs are a specific subset of helper T cells, inducing immune tolerance and moderating the intensity of immune reactions. Many autoimmune and neoplastic diseases are associated with T-reg impairment, favoring uncontrolled immune activation and attenuation of immune surveillance against tumor growth. An increase of polyclonal/nonclonal T cells in higher risk MDS, and a significant correlation of T-reg number, with the percentage of marrow blasts, the IPSS and progression to AML has been reported [37]. T-regs, characterized as CD4+ CD25high+FOXP3+ or CD4+CD25high+CD127low cells, were found increased in lower risk MDS but they were not correlated with any known disease feature or lab finding [38]. Investigation of the T-reg kinetics, function, and trafficking has revealed that in early MDS, peripheral blood and marrow T-regs are normal in number but dysfunctional, exhibiting lower CXCR4 expression and impaired marrow homing. In contrast, at late MDS or at leukemic transformation, T-regs increase and become functional and migrating. Effective treatment partially restores the number, but disease relapse is again associated with T-reg expansion. Thus, T-regs may share a pathophysiological role in MDS, since impaired sup‐ pressor function results in autoimmune phenomena, whereas in more advanced stages, their expansion favors clonal development and leukemic transformation [39]. It has been suggested that absolute number of a T-reg subpopulation, the "effector regulatory T cells," characterized as CD4+FOXP3+CD25+CD127lowCD45RA−CD27− cells, could be used as a prognostic factor in lower risk MDS predicting severity of anemia, AML transformation, and overall survival [40].

Other interesting T-cell subsets are the IL-17 producing helper T cells, the so-called Th17 cells and the Th22 cells. Th17 cells were found substantially increased in patients with lower risk MDS and their number was inversely correlated with that of T-regs. T-regs, although sup‐ pressive for other T-cell populations, do not affect Th17 cell number. Thus, the Th17/T-reg cell ratio has been found very high in lower risk disease and has been proposed as a marker of "effective" immunosuppression, high degree of apoptosis, and higher risk for autoimmunity, as well as an indicator for application of immunosuppressive treatment [41]. In contrast, helper T cells producing IL-22 (Th22 cells), involved in the pathogenesis of inflammatory reaction and autoimmunity, were found increased in patients with advanced MDS and their number was correlated with the mRNA levels of proinflammatory cytokines [42].

Finally, peripheral blood Tγδ lymphocytes, possessing a TCR with rearranged gamma/delta chains, and particularly Vγ9Vδ2 T cells, the major Tγδ-cell subset, which represent an important subpopulation for antitumor activity, were found reduced in patients with MDS and the reduction was greater in patients exhibiting autoimmune manifestations. Although Tγδ cells were not clonal, they reacted poorly to IL-2, and bromohalohydrin, a specific mitogen for these cells, induced mitogenic responses in only 60% of the MDS studied, unrelated to any specific disease feature. However, when activated, they exerted normal antileukemic effects against leukemic blasts. Therefore, the impaired number and function of this T-cell subpopulation may play a role in clonal expansion and disease progression of MDS [43].

#### **4.2. B-lymphocyte abnormalities**

Activated T lymphocytes do not belong to the dysplastic/leukemic clone and express HLA-DR, CD25, CD45RO, and CD57, but not CD28 and CD62L. This antigenic profile is independent of disease subtype, prognostic classification, kind of cytogenetic abnormality, or any other feature [33]. Interestingly, both patients with lower risk MDS and those with aplastic anemia have increased T-lymphocyte counts and B lymphocytopenia compared to patients with highrisk MDS and to controls, but lower risk MDS patients exhibit stronger and uniform Th1/Tc1 polarization than those with aplastic anemia [34]. Moreover, bone marrow NK T-cell infiltrates express the activated effector T-cell phenotype CD8+CD57+CD28−CD62L− and the NK Clectin-family receptors NKG2D and CD244. These infiltrates represent oligoclonal expansions of autoreactive T cells, as this can be demonstrated by TCR clonality assays and are more

MDS patients also exhibit impairment of delayed cutaneous T-cell hypersensitivity, as this can be demonstrated with various skin patch tests, challenging reaction to common antigens. Most importantly, they may lose immunologic memory against potentially important antigens, such as tuberculin and *clostr*. *tetani* anatoxin, the clinical consequence of which is unclear [36]. **Table 2** resumes the results from our study of skin reactions to the multitest CMI® in 54

Further understanding of the immune dysregulation of MDS was achieved through investi‐ gation of the regulatory T cells (T-regs). T-regs are a specific subset of helper T cells, inducing immune tolerance and moderating the intensity of immune reactions. Many autoimmune and neoplastic diseases are associated with T-reg impairment, favoring uncontrolled immune activation and attenuation of immune surveillance against tumor growth. An increase of polyclonal/nonclonal T cells in higher risk MDS, and a significant correlation of T-reg number, with the percentage of marrow blasts, the IPSS and progression to AML has been reported [37]. T-regs, characterized as CD4+ CD25high+FOXP3+ or CD4+CD25high+CD127low cells, were found increased in lower risk MDS but they were not correlated with any known disease feature or lab finding [38]. Investigation of the T-reg kinetics, function, and trafficking has revealed that in early MDS, peripheral blood and marrow T-regs are normal in number but dysfunctional, exhibiting lower CXCR4 expression and impaired marrow homing. In contrast, at late MDS or at leukemic transformation, T-regs increase and become functional and migrating. Effective treatment partially restores the number, but disease relapse is again associated with T-reg expansion. Thus, T-regs may share a pathophysiological role in MDS, since impaired sup‐ pressor function results in autoimmune phenomena, whereas in more advanced stages, their expansion favors clonal development and leukemic transformation [39]. It has been suggested that absolute number of a T-reg subpopulation, the "effector regulatory T cells," characterized as CD4+FOXP3+CD25+CD127lowCD45RA−CD27− cells, could be used as a prognostic factor in lower risk MDS predicting severity of anemia, AML transformation, and overall survival [40].

Other interesting T-cell subsets are the IL-17 producing helper T cells, the so-called Th17 cells and the Th22 cells. Th17 cells were found substantially increased in patients with lower risk MDS and their number was inversely correlated with that of T-regs. T-regs, although sup‐ pressive for other T-cell populations, do not affect Th17 cell number. Thus, the Th17/T-reg cell ratio has been found very high in lower risk disease and has been proposed as a marker of

prominently identified in the bone marrow than in the peripheral blood [35].

patients with MDS compared to 20 controls (unpublished data).

16 Myelodysplastic Syndromes

Although B lymphocytes in MDS patients do not demonstrate the spectrum of abnormalities detected in T cells, since their production and function is governed by T cells, their aberration actually reflects the functional integrity of T cells. Information for B lymphocytes is fewer and often conflicted. In many studies, decreased proportion and peripheral blood absolute B lymphocytopenia, frequently accompanied by hypogammaglobulinemia and recurrent infections has been reported [27, 44]. These findings are mostly confined to patients with lower risk disease and are associated with T-lymphocyte imbalance and reduced numbers of bone marrow B cells and B-cell precursors.

Analysis of the marrow CD34+ cell differentiation toward B lymphocytes in patients with lowrisk MDS has revealed low expression of B-lineage differentiation genes and reduced produc‐ tion of B-cell precursors, a finding proposed to be used as a hallmark of low-risk disease [45]. An additional factor contributing to B lymphocytopenia is that bone marrow B-, but not T lymphocytes, exhibit increased apoptosis, similar to that observed in nonlymphoid cells. Increased B-cell apoptosis could not be considered a clonal "property," since it was found neither in leukemic nor in normal marrows [44]. This was also confirmed on trephine biopsies of patients with high-risk MDS only, and the percentage of B lymphocytes was inversely correlated with prognosis [30]. Bone marrow flow cytometry analysis has shown increased proportion of CD34+CD45low B-cell precursors in patients with RA and RARS, and lower values in those with RAEB, whereas in AML B-cell precursors were not found at all, probably reflecting differentiation incapability of the CD34+ cells. Indeed, an inverse correlation of CD34+CD45low+ B cells with marrow blasts and a positive one with hemoglobin was found. Abnormal expression pattern of B-cell differentiation antigens, with hypoexpression of CD79α and TdT has also been reported, implying a possible role of the MDS marrow micro‐ environment in the maturation process of B lymphocytes [46].

Regarding the origin of B- and T lymphocytes, available data are again conflicting. In the majority of clonal studies, neither T- nor B lymphocytes or NK cells were found to originate from the dysplastic clone. Some studies have shown clonal origin of the B lymphocytes in a proportion of MDS patients, and in a Japanese study, the majority of patients with RA and those with immunological abnormalities exhibited clonal B lymphocytes [47]. Clonal origin was also found in 5% of the CD20+/CD22+ B cells of patients with trisomy 8. By using interphase FISH on sorted marrow cells, 13% of the CD5+CD19+ lymphocytes were clonal, implying that a part of the B lymphocytes in some patients may be clonal and that these cells may contribute to the manifestation of immune abnormalities [48].

B lymphocytes of MDS patients express low number of HLA-DR molecules (HLA class-II antigens) and are either deficient of EBV receptors or they carry abnormal Fcγ and C3d receptors, which cannot be used by EBV viral particles to enter and activate B cells. B-lym‐ phocyte cultures produce increased amounts of IL-6 and IL-10, following mitogenic stimula‐ tion. IL-6 is overproduced even without mitogenic stimuli. Finally, B lymphocytes of patients manifesting immune abnormalities have lower number of cell surface Fas ligand receptors, a finding possibly indicating their resistance to apoptosis.

Not surprisingly several quantitative serum γ-globulin and immunoglobulin abnormalities have been described in MDS patients, such as polyclonal hyper-γ-globulinemia, monoclonal M-spikes, or hypo-γ-globulinemia. Monoclonal components have been found significantly higher than in normal age-matched population [49]. It has been suggested that dysplastic monocytes might exert unspecific immune stimulation on B- and T lymphocytes through increased IL-1 production favoring the development of monoclonal B-cell populations and producing M-spikes.

Investigating the significance of serum protein electrophoresis in 158 patients, we noticed a normal pattern in 36% (mainly in RA and RARS) and only in 8% of CMML. A normal baseline pattern was associated with longer survival, independently of the IPSS and FAB classification. An acute phase reaction (alpha2-globulins >10 g/l) was seen in 17% at baseline, developed in additional 24% in the course of the disease, but in 80% of the patients transformed to AML and was associated with shorter survival. Hypo-γ-globulinemia was found in 6%, mainly RARS, was not related to frequent infections, and in RAEB it was associated with decreased marrow cellularity, deeper cytopenias, and longer survival. Polyclonal hyper-γ-globulinemia was found in 41% of patients (particularly RAEB-t, CMML) and monoclonal proteins in 16 cases (10%) more commonly in CMML and 2.5 times more frequently than in a control population of similar age. An additional 18% of the patients exhibited discrete M-components among polyclonal spectrum of γ-globulins. This finding has not yet been described and its significance is unclear [50].

#### **4.3. NK cell abnormalities**

Patients with MDS exhibit severe functional NK-cell impairment, but sometimes also numer‐ ical abnormalities. Cytotoxic NK-T cells, phenotypically characterized as CD3+CD8+CD16+, are usually normal or decreased but IFN-γ production is normal or increased. NK cells, characterized as CD3−CD8−CD11b+HNK1+ CD56+CD57+ cells, have been found normal, rarely decreased, but sometimes even increased [51]. The NK activity of MDS patients is almost always decreased compared to healthy controls [27, 51], although immunophenotypically NK cells are indistinguishable. In general, CD8+ T-cell function and the defective NK activity in MDS have been strongly and inversely correlated with bone marrow blast cell percentage, marrow cellularity, and serum sIL-2R levels [51]. Alloantigen- or mitogen-induced cellmediated cytotoxicity [27] as well as IFN-α and IL-2 production following NK cell activation is also impaired and the preincubation of NK cells with IFN-α may partially increase NK activity [52]. There are conflicting data regarding the origin of NK cells. By using FISH on FACS-sorted cells of patients with monosomy 7, monosomic signs in CD3−CD56+ cells were detected in 3 out of 4 [53]. In another study, between 20 and 50% but not all the NK cells were clonal, demonstrating a kind of "chimerism" by clonal and nonclonal NK cells in the majority of patients.

from the dysplastic clone. Some studies have shown clonal origin of the B lymphocytes in a proportion of MDS patients, and in a Japanese study, the majority of patients with RA and those with immunological abnormalities exhibited clonal B lymphocytes [47]. Clonal origin was also found in 5% of the CD20+/CD22+ B cells of patients with trisomy 8. By using interphase FISH on sorted marrow cells, 13% of the CD5+CD19+ lymphocytes were clonal, implying that a part of the B lymphocytes in some patients may be clonal and that these cells may contribute

B lymphocytes of MDS patients express low number of HLA-DR molecules (HLA class-II antigens) and are either deficient of EBV receptors or they carry abnormal Fcγ and C3d receptors, which cannot be used by EBV viral particles to enter and activate B cells. B-lym‐ phocyte cultures produce increased amounts of IL-6 and IL-10, following mitogenic stimula‐ tion. IL-6 is overproduced even without mitogenic stimuli. Finally, B lymphocytes of patients manifesting immune abnormalities have lower number of cell surface Fas ligand receptors, a

Not surprisingly several quantitative serum γ-globulin and immunoglobulin abnormalities have been described in MDS patients, such as polyclonal hyper-γ-globulinemia, monoclonal M-spikes, or hypo-γ-globulinemia. Monoclonal components have been found significantly higher than in normal age-matched population [49]. It has been suggested that dysplastic monocytes might exert unspecific immune stimulation on B- and T lymphocytes through increased IL-1 production favoring the development of monoclonal B-cell populations and

Investigating the significance of serum protein electrophoresis in 158 patients, we noticed a normal pattern in 36% (mainly in RA and RARS) and only in 8% of CMML. A normal baseline pattern was associated with longer survival, independently of the IPSS and FAB classification. An acute phase reaction (alpha2-globulins >10 g/l) was seen in 17% at baseline, developed in additional 24% in the course of the disease, but in 80% of the patients transformed to AML and was associated with shorter survival. Hypo-γ-globulinemia was found in 6%, mainly RARS, was not related to frequent infections, and in RAEB it was associated with decreased marrow cellularity, deeper cytopenias, and longer survival. Polyclonal hyper-γ-globulinemia was found in 41% of patients (particularly RAEB-t, CMML) and monoclonal proteins in 16 cases (10%) more commonly in CMML and 2.5 times more frequently than in a control population of similar age. An additional 18% of the patients exhibited discrete M-components among polyclonal spectrum of γ-globulins. This finding has not yet been described and its significance

Patients with MDS exhibit severe functional NK-cell impairment, but sometimes also numer‐ ical abnormalities. Cytotoxic NK-T cells, phenotypically characterized as CD3+CD8+CD16+, are usually normal or decreased but IFN-γ production is normal or increased. NK cells, characterized as CD3−CD8−CD11b+HNK1+ CD56+CD57+ cells, have been found normal, rarely decreased, but sometimes even increased [51]. The NK activity of MDS patients is almost always decreased compared to healthy controls [27, 51], although immunophenotypically NK

to the manifestation of immune abnormalities [48].

finding possibly indicating their resistance to apoptosis.

producing M-spikes.

18 Myelodysplastic Syndromes

is unclear [50].

**4.3. NK cell abnormalities**

Many groups investigated whether IFN-α treatment could induce blast/clonal cell clearance, through augmentation of the NK activity. In one study on 38 patients with RAEB, following 3-month treatment with IFN-α, NK activity and NK cell number and function was increased, but these alterations were not associated with any meaningful clinical response. NK cells exhibited normal tumor cell binding capacity, but inability of releasing cytotoxic factors, possibly suggesting intrinsic functional defects [52]. Another group did not confirm any quantitative defect and found normal expression of the activating receptors NKp46, NKp30, and NKG2D, but a depressed cytolytic activity. Incubation with IL-2 upregulated the NKp46 expression, but did not enhance NK-cell cytotoxicity but induced higher rate of apoptosis [53]. A strong correlation of the NK activity with higher IPSS, abnormal karyotype, excess of blasts and marrow hypercellularity, and downregulation of the NKG2D receptor has also been reported [54]. The Nordic MDS study Group showed that decreased expression of DNAM1 and NKG2D receptors on marrow NK cells was inversely correlated with blast percentage and suggested that DNAM1 plays a pivotal role in NK-mediated cell killing [55].

IL-12, alone or combined with IL-2, induces variable and unpredictable response to NK cells. Some patients (mainly with RA) exhibit a response closer to normal, while others respond poorly. The combination of IL-2 and IL-12 increases IFN-γ and TNF-α production in a synergistic way. IL-12 alone is not so stimulatory, and the combination of IL-2+IL-12 generates stimulation, similar to that obtained by IL-2 alone. Indeed, priming of peripheral blood mononuclear cells (PBMC) with IL-12 increased their cytotoxicity against autologous leukemic blasts to almost normal levels and significantly reduced WT1 mRNA expression, used as a marker of residual leukemic burden, except in patients with overt, high-bulk AML. Thus, *ex vivo* priming of cytotoxic NK-T and NK cells could be used as a tool, targeting residual disease, following systemic chemotherapy [56].

In a study from Düsseldorf, the authors recognized a small subgroup of high-risk patients, with almost absent peripheral blood NK cells, but intact populations of NK T cells. A larger subgroup with normal number but poor function of NK cells was characterized by reduced intracellular granzyme-B and perforin levels. This subgroup restored almost completely NKcell function, following mitogen or cytokine stimulation. NK cells were mainly immature but exhibited normal mature/activated (CD56bright+CD107+) immunophenotype and a restricted repertoire of KIR receptors. It is therefore suggested that the dysfunctional NK cells lead to inefficient/insufficient immune surveillance and clonal expansion [57]. The Pittsburgh Group reported different marrow frequencies of NK and NK T cells in MDS and AML. In MDS they did not find numerical impairment of the NK-cell population, but a significant decrease in mature CD56dimCD16+CD57bright cells, which had great prognostic significance for survival [58]. Other groups have reported increased intracellular granzyme-B levels in the NK cells of MDS patients [59].

#### **5. Serum cytokine profiles**

The immune-activated status of MDS patients lead to overproduction and elevated serum levels of many cytokines. We were the first group to report elevated serum soluble interleu‐ kin-2 receptors (sIL-2R) and tumor necrosis factor-α levels in 42 MDS patients confirming an abnormal immune stimulatory status. Although the difference in TNF-α levels between early and advanced MDS was not significant, patients with advanced MDS had significantly higher serum sIL-2R levels compared to those with early MDS [60]. *In vivo* treatment with rhGM-CSF or high-dose IL-3 further increases sIL-2R levels, which are associated with higher marrow cellularity and blast cell percentage, faster AML evolution, and shorter survival. These findings possibly reflect quantitative and qualitative abnormalities of the CD8+ and NK-cell subsets, resulting in ineffective T-B cell communication and impaired NK-cell function, since sIL-2R antagonizes the cellular receptor in IL-2 uptake, restricting T-cell activation [61]. sIL-2R levels are negatively correlated with T- and NK-cell counts and positively with adverse events occurring in the course of lower risk patients for whom sIL-2R levels are an independent adverse prognostic factor.

Serum IL-6 levels were found elevated in the majority of MDS patients and serum GM-CSF levels in less than half of them, although these cytokines were undetectable in normal subjects. Higher IL-6 concentrations were found in patients with advanced subtypes, were inversely correlated with the severity of the anemia and positively with peripheral blood and bone marrow blast cell percentages, and may increase further following chemotherapy. IL-6, IL-7, MCSF, TGFβ, and IL-1β are constitutively produced by marrow stromal cells of patients with MDS and AML, but not from stromal cells of normal subjects, and IL-6 gene transcription could be induced by exogenous addition of IL-1β confirming a cytokine network dysregulation [62]. Serum IL-8 levels were also found elevated, but they dropped under chemotherapy or during remission.

A Dutch group measured serum levels of seven cytokines in 75 MDS patients and found detectable levels of G-CSF in the majority of them, and increased IL-3 and IL-6 levels in a minority of patients but not in controls [63]. Serum TNF-α levels have been correlated with the severity of anemia, poor performance status, leucocytosis and monocytosis, higher β2 microglobulin and lower albumin levels, liver and renal impairment, and shorter survival [62– 64]. TNF-α levels <10 pg/ml have been associated with achievement of higher remission rate and longer PFS Progression Free and overall survival, whereas lower TNF-α and IL-1β levels could predict response to treatment with erythropoietin [64]. Thus, TNF-α represents the most important circulating and measurable cytokine, from the pathogenetic and the prognostic point of view. In general, serum levels of type-1 cytokines (IL-1β, IL-7, IL-8, IL-12, RANTES, and IFN-γ) [64, 65] are found elevated in lower risk MDS, whereas inhibitory factors (IL-10, sIL-2R) are elevated in higher risk disease.

The group of Mayo Clinic evaluated plasma levels of 30 different cytokines in 78 patients, and showed that although levels of 19 cytokines differed significantly from controls, in multivariate analysis, only levels of IL-6, IL-7, and CXCL10 had independent prognostic value for survival. Indeed, patients with normal levels of all these three cytokines had a median survival of 76 months compared to only 25 months for patients with elevated levels of at least one of them. For IL-6 levels in particular, a strong association with inferior leukemia-free survival, inde‐ pendent from other prognostic factors, was found [66].

Finally, a Spanish group, among other findings, demonstrated an inverse correlation of the CD3+, CD4+, and CD8+ populations with age, as well as an inverse correlation of serum IL-10 levels with the number of CD8+ cells, disease progression, and overall survival [67]. In another study, investigating the association of IL-10 gene polymorphisms with the development and the features of MDS, the highly IL-10-expressing genotype *-592 CC* was associated with more severe anemia and poorer survival compared to non-IL10-expressing genotypes, thus con‐ firming a significant prognostic role for IL-10 [68].

#### **6. Functional immunoregulatory abnormalities of T lymphocytes**

#### **6.1. Mixed lymphocyte reactions (MLRs): basic information**

reported different marrow frequencies of NK and NK T cells in MDS and AML. In MDS they did not find numerical impairment of the NK-cell population, but a significant decrease in mature CD56dimCD16+CD57bright cells, which had great prognostic significance for survival [58]. Other groups have reported increased intracellular granzyme-B levels in the NK cells of MDS

The immune-activated status of MDS patients lead to overproduction and elevated serum levels of many cytokines. We were the first group to report elevated serum soluble interleu‐ kin-2 receptors (sIL-2R) and tumor necrosis factor-α levels in 42 MDS patients confirming an abnormal immune stimulatory status. Although the difference in TNF-α levels between early and advanced MDS was not significant, patients with advanced MDS had significantly higher serum sIL-2R levels compared to those with early MDS [60]. *In vivo* treatment with rhGM-CSF or high-dose IL-3 further increases sIL-2R levels, which are associated with higher marrow cellularity and blast cell percentage, faster AML evolution, and shorter survival. These findings possibly reflect quantitative and qualitative abnormalities of the CD8+ and NK-cell subsets, resulting in ineffective T-B cell communication and impaired NK-cell function, since sIL-2R antagonizes the cellular receptor in IL-2 uptake, restricting T-cell activation [61]. sIL-2R levels are negatively correlated with T- and NK-cell counts and positively with adverse events occurring in the course of lower risk patients for whom sIL-2R levels are an independent

Serum IL-6 levels were found elevated in the majority of MDS patients and serum GM-CSF levels in less than half of them, although these cytokines were undetectable in normal subjects. Higher IL-6 concentrations were found in patients with advanced subtypes, were inversely correlated with the severity of the anemia and positively with peripheral blood and bone marrow blast cell percentages, and may increase further following chemotherapy. IL-6, IL-7, MCSF, TGFβ, and IL-1β are constitutively produced by marrow stromal cells of patients with MDS and AML, but not from stromal cells of normal subjects, and IL-6 gene transcription could be induced by exogenous addition of IL-1β confirming a cytokine network dysregulation [62]. Serum IL-8 levels were also found elevated, but they dropped under chemotherapy or during

A Dutch group measured serum levels of seven cytokines in 75 MDS patients and found detectable levels of G-CSF in the majority of them, and increased IL-3 and IL-6 levels in a minority of patients but not in controls [63]. Serum TNF-α levels have been correlated with the severity of anemia, poor performance status, leucocytosis and monocytosis, higher β2 microglobulin and lower albumin levels, liver and renal impairment, and shorter survival [62– 64]. TNF-α levels <10 pg/ml have been associated with achievement of higher remission rate and longer PFS Progression Free and overall survival, whereas lower TNF-α and IL-1β levels could predict response to treatment with erythropoietin [64]. Thus, TNF-α represents the most important circulating and measurable cytokine, from the pathogenetic and the prognostic

patients [59].

20 Myelodysplastic Syndromes

**5. Serum cytokine profiles**

adverse prognostic factor.

remission.

A basic property of the immunocompetent cells is the recognition of the "self" and the orchestration of an immune response against the "nonself" or the "altered self," and when selfrecognition is impaired, an autoimmune disorder emerges. An initial interpretation for the frequent autoimmune disorders and other immune abnormalities of MDS patients was that they might probably reflect clonal origin of B- and T lymphocytes. Later, however, it was demonstrated that, in the majority of cases, B- and T lymphocytes are nonclonal, but Tlymphocyte abnormalities may influence disease course. Various T-lymphocyte subsets exert complex immunoregulatory activities on other T-cell populations, B lymphocytes, and monocytes. Mixed lymphocyte cultures are performed with the coculture of a pure T-cell population (responder cells), upon which a kinetically inactive, but cell-surface intact, non-T cell population (stimulant cells) affects, thus generating a mixed lymphocyte reaction. MLRs represent dynamic *in vitro* models for the study of various cellular interactions and of the immunoregulatory mechanisms developed between different immunocompetent cell popu‐ lations. When the stimulant and the responder cell population stem from the same subject, the model is called *autologous MLR* (AMLR), whereas when the stimulant population stems from another subject the model is called *allogeneic MLR* (Allo-MLR). AMLR and Allo-MLR constitute practical tools for the investigation of various diseases and conditions with an underlying immune-based pathogenesis or pathophysiology.

The proliferative reaction (MLR) is mediated through recognition of structural antigenic domains of the cell surface of non-T cells, and particularly HLA class-II antigens. The stimu‐ lating capacity of the non-T-cell population is abrogated when cell membrane structure is destroyed, either following mechanical stress or treatment with proteolytic enzymes. More‐ over, the stimulatory capacity is not a soluble factor and non-T- or B-lymphocyte supernatants do not retain any stimulatory activity on T lymphocytes [69], whereas preincubation of the non-T-cell population with anti-HLA-DR monoclonal antibodies completely abrogates the AMLR and substantially depresses the Allo-MLR. The stimulatory potential of other mem‐ brane determinants on the MLRs was identified in a similar way. Such molecules are HLA class I (HLA-A, -B, and -C) for the Allo-MLR, CD3-Ti complex for any type of MLR, and probably additional minor antigenic determinants of the MHC. Stimulating capacity of the non-T-cell population is dependent on the various mononuclear cell constituents included. B lymphocytes are stronger stimulants than NK cells and null lymphocytes. Activated B lymphocytes, surface IgM(+) B lymphocytes, and B lymphoblasts are better stimulants than resting- and IgM(−) B lymphocytes, and this property is independent of their content in EBV-DNA or the origin from a mitogen-enriched culture. The role of monocytes is contradictory, since inactive monocytes enhance autologous reactivity, whereas the admixture of monocytes in the responder T-cell population results in severe impairment of both AMLR and Allo-MLR.

MLRs share the characteristic features of an orchestrated immune reaction showing immuno‐ logic memory and specificity. When T lymphocytes, previously exposed to autologous non-T cells and obtained the seventh day of culture, are re-exposed to the same non-T-cell population, they demonstrate their peak proliferation earlier, on the third day of culture (secondary AMLR), thanks to previously engrafted immunologic memory. The same has been confirmed for the Allo-MLR, because when in the secondary culture the allogenic stimuli are different, then different responder T-cell population is activated and the reaction shows the kinetic of the primary MLR. There are different autoreactive and alloreactive T-cell populations. The number of alloreactive T cells is 5–40 times higher than autoreactive, and represents 1/400– 1/150 of the total peripheral blood T lymphocytes, whereas autoreactive constitute 1/5000– 1/2200 of them.

The basic function of the MLRs is the production of suppressor "activity" or of suppressor/ cytotoxic T cells. The main part of the responder population are CD4+ helper/inducer T lymphocytes and treatment of this population with an anti-CD4 monoclonal antibody, practically abrogates all types of MLR. Conversely, treatment with an anti-CD8 antibody quantitatively decreases the strength of both types of MLR. Thus, from the relative content of the two major T-cell subpopulations AMLR has two different phases. Responder CD4+ T lymphocytes undergo a proliferative reaction upon sensation of autologous signals (self-MHC antigens: autoreactive T cells). CD4+ cell proliferative reaction is peaked on the third and fourth day of the culture, when helper T-cell population dominates. This reaction is followed by a secondary activation of the suppressor CD8+ T cells, which is quantitatively stronger, is peaked on the sventh and eighth day of the culture and inhibits any further proliferation of the autoreactive T cells. This serially fulfilled lymphocyte reaction is mediated through the production of IL-2 by the CD4+ T cells. In the Allo-MLR the responder population (alloreactive T cells) is activated through the recognition of the MHC alloantigens and is consisted of both helper and suppressor T lymphocytes. Allo-MLR is always stronger than AMLR. Deficiency of AMLR and of Allo-MLR has been reported in various diseases and conditions, a list of which is provided in **Table 3**.

lating capacity of the non-T-cell population is abrogated when cell membrane structure is destroyed, either following mechanical stress or treatment with proteolytic enzymes. More‐ over, the stimulatory capacity is not a soluble factor and non-T- or B-lymphocyte supernatants do not retain any stimulatory activity on T lymphocytes [69], whereas preincubation of the non-T-cell population with anti-HLA-DR monoclonal antibodies completely abrogates the AMLR and substantially depresses the Allo-MLR. The stimulatory potential of other mem‐ brane determinants on the MLRs was identified in a similar way. Such molecules are HLA class I (HLA-A, -B, and -C) for the Allo-MLR, CD3-Ti complex for any type of MLR, and probably additional minor antigenic determinants of the MHC. Stimulating capacity of the non-T-cell population is dependent on the various mononuclear cell constituents included. B lymphocytes are stronger stimulants than NK cells and null lymphocytes. Activated B lymphocytes, surface IgM(+) B lymphocytes, and B lymphoblasts are better stimulants than resting- and IgM(−) B lymphocytes, and this property is independent of their content in EBV-DNA or the origin from a mitogen-enriched culture. The role of monocytes is contradictory, since inactive monocytes enhance autologous reactivity, whereas the admixture of monocytes in the responder T-cell population results in severe impairment of both AMLR and Allo-MLR.

MLRs share the characteristic features of an orchestrated immune reaction showing immuno‐ logic memory and specificity. When T lymphocytes, previously exposed to autologous non-T cells and obtained the seventh day of culture, are re-exposed to the same non-T-cell population, they demonstrate their peak proliferation earlier, on the third day of culture (secondary AMLR), thanks to previously engrafted immunologic memory. The same has been confirmed for the Allo-MLR, because when in the secondary culture the allogenic stimuli are different, then different responder T-cell population is activated and the reaction shows the kinetic of the primary MLR. There are different autoreactive and alloreactive T-cell populations. The number of alloreactive T cells is 5–40 times higher than autoreactive, and represents 1/400– 1/150 of the total peripheral blood T lymphocytes, whereas autoreactive constitute 1/5000–

The basic function of the MLRs is the production of suppressor "activity" or of suppressor/ cytotoxic T cells. The main part of the responder population are CD4+ helper/inducer T lymphocytes and treatment of this population with an anti-CD4 monoclonal antibody, practically abrogates all types of MLR. Conversely, treatment with an anti-CD8 antibody quantitatively decreases the strength of both types of MLR. Thus, from the relative content of the two major T-cell subpopulations AMLR has two different phases. Responder CD4+ T lymphocytes undergo a proliferative reaction upon sensation of autologous signals (self-MHC antigens: autoreactive T cells). CD4+ cell proliferative reaction is peaked on the third and fourth day of the culture, when helper T-cell population dominates. This reaction is followed by a secondary activation of the suppressor CD8+ T cells, which is quantitatively stronger, is peaked on the sventh and eighth day of the culture and inhibits any further proliferation of the autoreactive T cells. This serially fulfilled lymphocyte reaction is mediated through the production of IL-2 by the CD4+ T cells. In the Allo-MLR the responder population (alloreactive T cells) is activated through the recognition of the MHC alloantigens and is consisted of both helper and suppressor T lymphocytes. Allo-MLR is always stronger than AMLR. Deficiency

1/2200 of them.

22 Myelodysplastic Syndromes


**Table 3.** Diseases with an abnormal autologous mixed lymphocyte reaction.

Besides the immunoregulatory cell circuits, generated following "autorecognition" in the AMLR, helper and suppressor T lymphocytes exert regulatory function in normal hemato‐ poiesis. T lymphocytes obtained at the early phase of the AMLR (third day) have promoting activity on the formation of early (bursts) and late erythroid colonies (CFU-E) and this activity is similar to that obtained by PHA-activated T lymphocytes. This activity, initially termed *Burst Promoting Activity*, is attributed to the production of various hematopoietic cytokines and particularly interleukin-3. Following the development of suppressor activity for damp‐ ening autologous reaction on the seventh culture day, this activity also induces suppression of development of immature erythropoietic and other progenitor cells, similar to that gener‐ ated from activated lymphocytes following prolonged antigenic stimulation, as this happens in chronic infections, inflammatory conditions, connective tissue diseases, and in aplastic anemia. In these situations activated suppressor T lymphocytes produce suppressive cyto‐ kines, and particularly but not exclusively IFN-γ.

Cytotoxic activity, generated in the MLRs, is also directed against autologous and allogeneic B lymphocytes, monocytes, and cells with an altered antigenic profile, either neoplastic or not. Generation of cytotoxic activity against tumor cells as a result of immune activation is of tremendous clinical significance. In AMLR and Allo-MLR the neoplastic cells may represent the stimulant cell population, whereas responder populations might be both the suppressor/ cytotoxic CD3+CD16+ NK-T cells and the CD3-CD16+CD56+ NK cells. Activation of these populations results in increased proliferation and the adoption of an activated profile. Thus, cytotoxic T cells generated in AMLR may play an important role in antitumor surveillance [70].

#### **6.2. Autologous and allogeneic MLRs of patients with myelodysplastic syndromes**

AMLR and Allo-MLR were found significantly reduced on 12 MDS patients and the amount of IL-2 produced during these reactions was severely depressed. Exogenous addition of IL-2 partially restored the strength of the reactions, which, however, continued to be substantially reduced compared to normal controls. To investigate which cell population was primarily affected, the authors compared the strength of Allo-MLR by using allogeneic non-T cells from both normal controls and other MDS patients against T cells from MDS patients. They also tested Allo-MLR of T lymphocytes from healthy controls against non-T cells obtained either from normal subjects or from MDS patients. Allo-MLR of T cells from MDS patients was substantially improved with the use of normal allogeneic non-T cells, but did not reach the normal range. Conversely, Allo-MLR of T cells from normal subjects was severely deficient when stimulant non-T cells had been obtained from MDS patients compared to the reaction against non-T cells from normal subjects. Therefore, it appears that in MDS there is an impaired stimulating capacity of the non-T-cell population consisting of B lymphocytes, monocytes, and immature myeloid cells [26]. Almost concurrently the presence of "leukemia-inhibitory activity" (LIA) of peripheral blood non-T cells of MDS patients mainly obvious in patients with an excess of blasts, but also in some patients without an excess of blasts (RA-RARS) was reported. Moreover, the majority of the patients without an excess of blasts, whose serum contained LIA, evolved quickly to RAEB/AML. This "activity" of higher risk MDS patients could be eluted from culture of PBMC in FCS-enriched media with the addition of GM-CSF and IL-4 [71]. Cells responsible for the induction of suppression/inhibition of cell growth are clonal macrophages, transformed in culture to "giant macrophages" or dendritic cells. The mediator of suppression was a soluble factor, other than IFN-γ or TNF-α, identified as acidic isoferritin [72].

activity on the formation of early (bursts) and late erythroid colonies (CFU-E) and this activity is similar to that obtained by PHA-activated T lymphocytes. This activity, initially termed *Burst Promoting Activity*, is attributed to the production of various hematopoietic cytokines and particularly interleukin-3. Following the development of suppressor activity for damp‐ ening autologous reaction on the seventh culture day, this activity also induces suppression of development of immature erythropoietic and other progenitor cells, similar to that gener‐ ated from activated lymphocytes following prolonged antigenic stimulation, as this happens in chronic infections, inflammatory conditions, connective tissue diseases, and in aplastic anemia. In these situations activated suppressor T lymphocytes produce suppressive cyto‐

Cytotoxic activity, generated in the MLRs, is also directed against autologous and allogeneic B lymphocytes, monocytes, and cells with an altered antigenic profile, either neoplastic or not. Generation of cytotoxic activity against tumor cells as a result of immune activation is of tremendous clinical significance. In AMLR and Allo-MLR the neoplastic cells may represent the stimulant cell population, whereas responder populations might be both the suppressor/ cytotoxic CD3+CD16+ NK-T cells and the CD3-CD16+CD56+ NK cells. Activation of these populations results in increased proliferation and the adoption of an activated profile. Thus, cytotoxic T cells generated in AMLR may play an important role in antitumor surveillance [70].

**6.2. Autologous and allogeneic MLRs of patients with myelodysplastic syndromes**

AMLR and Allo-MLR were found significantly reduced on 12 MDS patients and the amount of IL-2 produced during these reactions was severely depressed. Exogenous addition of IL-2 partially restored the strength of the reactions, which, however, continued to be substantially reduced compared to normal controls. To investigate which cell population was primarily affected, the authors compared the strength of Allo-MLR by using allogeneic non-T cells from both normal controls and other MDS patients against T cells from MDS patients. They also tested Allo-MLR of T lymphocytes from healthy controls against non-T cells obtained either from normal subjects or from MDS patients. Allo-MLR of T cells from MDS patients was substantially improved with the use of normal allogeneic non-T cells, but did not reach the normal range. Conversely, Allo-MLR of T cells from normal subjects was severely deficient when stimulant non-T cells had been obtained from MDS patients compared to the reaction against non-T cells from normal subjects. Therefore, it appears that in MDS there is an impaired stimulating capacity of the non-T-cell population consisting of B lymphocytes, monocytes, and immature myeloid cells [26]. Almost concurrently the presence of "leukemia-inhibitory activity" (LIA) of peripheral blood non-T cells of MDS patients mainly obvious in patients with an excess of blasts, but also in some patients without an excess of blasts (RA-RARS) was reported. Moreover, the majority of the patients without an excess of blasts, whose serum contained LIA, evolved quickly to RAEB/AML. This "activity" of higher risk MDS patients could be eluted from culture of PBMC in FCS-enriched media with the addition of GM-CSF and IL-4 [71]. Cells responsible for the induction of suppression/inhibition of cell growth are clonal macrophages, transformed in culture to "giant macrophages" or dendritic cells. The

kines, and particularly but not exclusively IFN-γ.

24 Myelodysplastic Syndromes

We investigated the MLRs in 20 MDS patients in paired experiments with sex- and agematched controls at baseline, before the administration of any interventional treatment. To express the strength of reactions we used the *Stimulation Index*, i.e., the ratio of the incorporated 3 H-thymidine in the MLR divided by the incorporated 3 H-thymidine in an unstimulated culture of equal number of purified CD3+ T lymphocytes. Patients with MDS exhibited severely impaired AMLR in all experiments with a median value almost half as that of the controls, without overlapping values, and the difference between the two groups was statis‐ tically very significant (*p* < 0.000001). Patients with RAEB showed that the most attenuated reactions were significantly weaker than the remaining patients [73]. Cumulative results are shown in **Table 4**.


**Table 4.** Results of the autologous mixed lymphocyte reaction in patients with MDS (counts per min and stimulation index, S.I.) Bold letters/numbers indicate statistically significant differences.

To evaluate the capability of the stimulant cell population, Allo-MLR was performed against non-T cells originating either from another MDS patient or from a healthy control. Moreover, to evaluate the capability of the responder cells, Allo-MLR of the healthy controls was performed against non-T cells from MDS patients or from other controls. In all cases, Allo-MLR against normal non-T cells was substantially higher, and on average threefold as strong as AMLR and Allo-MLR against "dysplastic" non-T cells was weaker, but always stronger than AMLR of the same person. The difference between these two types of controls' Allo-MLR was significant (S.I.: 7.90 ± 0.89 versus 14.12 ± 1.59, *p* = 0.0035, unpublished data). When compared to controls, Allo-MLR of MDS patients was significantly impaired in all comparisons (S.I.: 4.53 ± 0.41 versus 14.12 ± 1.59, *p* = 0.000014, unpublished data). Significant difference was maintained in the comparison of Allo-MLR between healthy controls and patients with RA, RARS, and RAEB separately, whereas CMML patients exhibited the less, and RAEB patients the most attenuated reactions, significantly weaker than the remaining MDS. In paired analysis, alloreactivity of MDS patients was always weaker than that of the corresponding control and the ratio Patient's Allo-MLR/Control's Allo-MLR was always <1 (median 0.36, range 0.06–0.58). Among MDS patients, "dysplastic" origin of the non-T cells did not further impair the already depressed alloreactivity. However, even in this Allo-MLR the difference between patients and controls was still significant (S.I.: 3.64 ± 0.21 versus 7.90 ± 0.89, *p* = 0.026) [73]. Results of the Allo-MLR are shown in **Table 5**.


**Table 5.** Allogeneic mixed lymphocyte reaction in patients with myelodysplastic syndromes – counts per min and stimulation index (S.I.) Bold letters/numbers indicate statistically significant differences.

Similar results were obtained by the Czech group who found significantly decreased MLRs with lower TNF-α and IFN-γ production in the supernatants of patients with RA compared to the MLRs of RARS patients. They also found less affected the allo-MLR against normal non-T cells, and identified as more defective the second (effector) phase of the reaction [74]. Therefore, in the MLRs of MDS patients there is an impairment of both the responder (T cells) and the stimulant population (non-T cells). The responder population reacts poorly to autologous and allogeneic stimuli and exhibits a profile of immune tolerance, which is clearer in the high-risk patients. Moreover, the stimulant population provides insufficient stimuli for reaction to the T cells, since it also depresses the alloreactivity of normal T lymphocytes. The possible, if any, clinical consequences of these findings are practically unknown or remain only speculative.

#### **6.3. Pathogenesis of immune dysregulation in MDS: immune abnormalities or immune adaptation?**

#### *6.3.1. Autoreactivity against the clone: Autologous progenitor cell/T-lymphocyte reaction (APLR)*

"Inhibitory activity" derived from serum and PBMC culture's supernatants of MDS patients has earlier been described and associated with poor prognosis [71]. Normal PBMC inhibit autologous hematopoietic cell colony formation in short-term cultures, as did also PBMC of patients with RA, but they induced a clear inhibitory activity later on day 10. Responsible cells are probably cytotoxic T and NK cells, which may react against the clone and suppress the growth of clonal cells at early stages of the disease. If this suppressor function develops early and is effective, clonal growth may be arrested. Nevertheless, NK cells of MDS patients usually exhibit impaired function and sometimes are clonal. However, since suppressor activity is achieved through various soluble cytokines and mainly through TNF-α, it is not quite specific and may also affect nonclonal cells, resulting in hematopoietic suppression, as this is observed in hypoplastic MDS and aplastic anemia. Indeed, lymphocyte culture supernatants from MDS patients exert suppressive activity on the growth of normal hematopoietic progenitors [75].

impair the already depressed alloreactivity. However, even in this Allo-MLR the difference between patients and controls was still significant (S.I.: 3.64 ± 0.21 versus 7.90 ± 0.89, *p* = 0.026)

**Normal non-T cells Dysplastic non-T cells FAB Group N × ±SEM p N × ±SEM p cpm S.I. cpm S.I.** RA 4 3562 ± 334 4.79 ± 0.64 **0.020** 3 2532 ± 407 3.01 ± 0.23 n.s. RAS 5 4569 ± 702 5.86 ± 0.87 **0.021** 4 3859 ± 685 4.89 ± 1.16 n.s. RAEB 8 2643 ± 328 3.04 ± 0.25 **< 0.000** 7 1865 ± 563 2.21 ± 0.49 n.s. CMML 3 4215 ± 695 5.93 ± 0.62 0.070 1 3380 4.47 – All Pts 20 3544 ± 312 4.53 ± 0.41 **< 0.0001** 15 2697 ± 389 3.63 ± 0.21 **0.040**

Controls 20 11,355 ± 1459 14.12 ± 1.59 12 6558 ± 869 7.90 ± 0.89

RAEB vs. all other MDS (S.I.) 3.04 ± 0.29 **<0.001** 5.52 ± 0.47 **0.001**

Similar results were obtained by the Czech group who found significantly decreased MLRs with lower TNF-α and IFN-γ production in the supernatants of patients with RA compared to the MLRs of RARS patients. They also found less affected the allo-MLR against normal non-T cells, and identified as more defective the second (effector) phase of the reaction [74]. Therefore, in the MLRs of MDS patients there is an impairment of both the responder (T cells) and the stimulant population (non-T cells). The responder population reacts poorly to autologous and allogeneic stimuli and exhibits a profile of immune tolerance, which is clearer in the high-risk patients. Moreover, the stimulant population provides insufficient stimuli for reaction to the T cells, since it also depresses the alloreactivity of normal T lymphocytes. The possible, if any, clinical consequences of these findings are practically unknown or remain only

**6.3. Pathogenesis of immune dysregulation in MDS: immune abnormalities or immune**

*6.3.1. Autoreactivity against the clone: Autologous progenitor cell/T-lymphocyte reaction (APLR)*

"Inhibitory activity" derived from serum and PBMC culture's supernatants of MDS patients has earlier been described and associated with poor prognosis [71]. Normal PBMC inhibit autologous hematopoietic cell colony formation in short-term cultures, as did also PBMC of patients with RA, but they induced a clear inhibitory activity later on day 10. Responsible cells are probably cytotoxic T and NK cells, which may react against the clone and suppress the growth of clonal cells at early stages of the disease. If this suppressor function develops early and is effective, clonal growth may be arrested. Nevertheless, NK cells of MDS patients usually

**Table 5.** Allogeneic mixed lymphocyte reaction in patients with myelodysplastic syndromes – counts per min and

stimulation index (S.I.) Bold letters/numbers indicate statistically significant differences.

speculative.

**adaptation?**

[73]. Results of the Allo-MLR are shown in **Table 5**.

26 Myelodysplastic Syndromes

This form of cytotoxicity was also identified in high-risk MDS and in AML, and was attributed to possible infection of leukemic cells by an oncogenic virus. However, viral infection is not necessary for the generation of an immune reaction, since clonal cells contain and sometimes express on their membrane many abnormal or mutated proteins, possibly representing neoantigens capable to induce lymphocytotoxic reactions by CD8+ T cells. Autoantigens are hardly found in MDS and may only be speculative but have been identified in some other marrow failure syndromes, such as aplastic anemia and paroxysmal nocturnal hemoglobinu‐ ria. As possible antigens, the Wilms Tumor protein (WT1), moesin, a cytoskeleton protein, KIF20B (kinesin), desmoplakin, and proteinase-3, an enzyme of blast-cell granules, have been indicated [76]. T lymphocytes of some MDS/AML patients stimulated *in vitro* with WT1 and proteinase-3 were polarized toward TH1 direction with the production of IFN-γ and the enrichment of their cytoplasm with granzyme B [77]. Moreover, patients expressing defined proteinase-3 aplotype generate stronger allogeneic lymphocytotoxic reactions following allogeneic hematopoietic stem-cell transplantation (GVL effect).

A challenging hypothesis is that the adaptive immunity may rather "react" than be impaired following various cellular interactions, and this immune "reaction," or at least such cellular interactions, might be a part of the pathogenesis of MDS. This "reaction" also may represent a defensive mechanism of the immune system against the dysplastic/neoplastic clone and is orchestrated specifically against clonal bone marrow cells. Specific CD8+ suppressor/cytotoxic T cells recognizing progenitor cells with trisomy 8 have been identified in MDS patients with this abnormality. Clonal inhibition is achieved via MHC class I recognition and through induction of FAS-mediated apoptosis [78]. The possible contributing role of an altered marrow microenvironment in the development of such immune alterations is also tempting.

The presence of increased number of immunocompetent T lymphocytes with an activated cytotoxic immunophenotype CD8+CD25+CD28-CD57+ has been reported in the marrows of patients with aplastic anemia and MDS. These cells do not to directly influence the severity of peripheral blood cytopenias [79]. In our study on 41 patients, the percentage of activated marrow suppressor/cytotoxic T lymphocytes was inversely correlated with marrow cellularity and blast cell percentage, and positively with Fas antigen expression on CD34+ clonal pro‐ genitor cells [80]. The cytotoxic reaction against marrow CD34+ cells of MDS patients has a well-defined signal transduction pathway in the T cells and can be augmented *in vitro* with the exogenous addition of IL-2 [81]. The strength of this reaction has not been associated with any TNF-α-, IL-10-, or lymphotoxin gene polymorphism, although as it is well-known that these polymorphisms appear to influence the severity of acute GVHD, following allogeneic hematopoietic stem-cell transplantation [82].

We also investigated the behavior of the clonal CD34+ progenitors as stimulant population in mixed cultures with autologous T lymphocytes as responder cells, in other words the immune reaction when T cells are in close contact with clonal stem cells. We compared this type of reaction (autologous progenitor cell mixed lymphocyte reaction—APLR) with the classical types of MLRs. APLR reflects the strength of the immune reaction against the clone in a background of established relative immune tolerance. We tested APLR in 20 MDS patients and 10 healthy controls. We noticed significant differences in the strength of the reaction between patients and controls, as well as between the various subtypes of MDS. Results are shown in **Table 6**. Among normal subjects APLR was rather a mild proliferative reaction, less than half strong as AMLR and about six- to sevenfold weaker than Allo-MLR. Among MDS patients, APLR was significantly stronger than in controls (*p* = 0.048, unpublished data, see **Table 6**). Stimulation index ranged between 1.8 and 26.0 in patients, and between 1.4 and 3.0 in controls. Thus, APLR was the only MLR in which MDS patients exhibited stronger reactions than controls and with high variability [83]. In particular, patients without excess of blasts had APLR similar to normal subjects, whereas patients with RAEB showed significantly stronger reactions. Specifically, a subgroup of four RAEB patients exhibited very strong reactions with a SI >10, significantly higher than the remaining MDS patients, although the same patients developed weak responses against autologous and allogeneic non-T cell stimuli. As mentioned earlier, in all healthy controls the ratio APLR/AMPR was always <1. In patients with RA or RARS this ratio was around 1 but in some of them higher than 1, whereas in patients with RAEB, APLR/AMLR ratio was substantially higher than 1. Thus, the three subject groups tested with MLRs (low-risk MDS, high-risk MDS, and controls) could be compartmentalized in three different areas in the plot (see **Figure 1**).


**Table 6.** Autologous progenitor cell mixed lymphocyte reaction (APLR) Counts per min and stimulation index Bold letters/numbers indicate statistically significant differences.

Our results have been confirmed by Chamuleau et al., who demonstrated increased non-MHCrestricted autologous cytotoxicity against clonal marrow precursors in eight patients with lower risk MDS, possibly indicating immune surveillance against clonal expansion although they have not provided clinical data on its significance [59]. Suppression may not be restricted against the dysplastic clone and may also affect nonclonal (normal) hematopoiesis. Lympho‐ cyte-depleted long-term bone marrow cultures from patients with lower risk MDS also generated some nonclonal hematopoietic colony growth, which was abrogated when T lymphocytes were present in the culture system [82]. Autologous lymphocytes were particu‐ larly cytotoxic in patients with hypoplastic MDS, trisomy 8, or bearing the DR15 allele [84]. The suppressive role of autologous T lymphocytes has been very nicely demonstrated in a patient with MDS and cyclic hematopoiesis, in whom, the percentage of marrow CD3+ lymphocytes was inversely correlated with neutrophil and platelet count during the various phases of ineffective hematopoiesis [85].

reaction when T cells are in close contact with clonal stem cells. We compared this type of reaction (autologous progenitor cell mixed lymphocyte reaction—APLR) with the classical types of MLRs. APLR reflects the strength of the immune reaction against the clone in a background of established relative immune tolerance. We tested APLR in 20 MDS patients and 10 healthy controls. We noticed significant differences in the strength of the reaction between patients and controls, as well as between the various subtypes of MDS. Results are shown in **Table 6**. Among normal subjects APLR was rather a mild proliferative reaction, less than half strong as AMLR and about six- to sevenfold weaker than Allo-MLR. Among MDS patients, APLR was significantly stronger than in controls (*p* = 0.048, unpublished data, see **Table 6**). Stimulation index ranged between 1.8 and 26.0 in patients, and between 1.4 and 3.0 in controls. Thus, APLR was the only MLR in which MDS patients exhibited stronger reactions than controls and with high variability [83]. In particular, patients without excess of blasts had APLR similar to normal subjects, whereas patients with RAEB showed significantly stronger reactions. Specifically, a subgroup of four RAEB patients exhibited very strong reactions with a SI >10, significantly higher than the remaining MDS patients, although the same patients developed weak responses against autologous and allogeneic non-T cell stimuli. As mentioned earlier, in all healthy controls the ratio APLR/AMPR was always <1. In patients with RA or RARS this ratio was around 1 but in some of them higher than 1, whereas in patients with RAEB, APLR/AMLR ratio was substantially higher than 1. Thus, the three subject groups tested with MLRs (low-risk MDS, high-risk MDS, and controls) could be compartmentalized in three

**FAB Group N cpm (Mean ± SEM) Stim. Index (Mean ± SEM) p APLR pt/APLR control**

**Table 6.** Autologous progenitor cell mixed lymphocyte reaction (APLR) Counts per min and stimulation index Bold

Our results have been confirmed by Chamuleau et al., who demonstrated increased non-MHCrestricted autologous cytotoxicity against clonal marrow precursors in eight patients with lower risk MDS, possibly indicating immune surveillance against clonal expansion although they have not provided clinical data on its significance [59]. Suppression may not be restricted against the dysplastic clone and may also affect nonclonal (normal) hematopoiesis. Lympho‐ cyte-depleted long-term bone marrow cultures from patients with lower risk MDS also

RA 4 2334 ± 382 2.88 ± 0.41 0.173 1.21 ± 0.11 RAS 5 2365 ± 385 2.77 ± 0.18 0.109 1.34 ± 0.13 RAEB 8 7502 ± 1194 10.35 ± 2.46 **0.001** 3.92 ± 0.74 CMML 3 3488 ± 556 4.55 ± 0.25 **<0.001** 1.43 ± 0.12 All patients 20 4582 ± 735 6.09 ± 1.27 **0.048** 2.39 ± 0.85

Other MDS 12 3.26 ± 0.95 **0.004**

different areas in the plot (see **Figure 1**).

28 Myelodysplastic Syndromes

Controls 10 1866 ± 308 2.21 ± 0.31 RAEB 8 7502 ± 1194 10.35 ± 2.46

letters/numbers indicate statistically significant differences.

**Figure 1.** Correlation of AMLR to APLR in each subject tested with MLRs. By correlating AMLR to APLR an almost complete, discrete compartmentalization of the three main subject groups was found. Normal controls exhibited high‐ er AMLR than APLR, patients with lower risk MDS had lower, both types of MLRs, whereas patients with an excess of marrow blasts showed impaired AMLR and very increased APLR.

#### **6.4. The role of clonal hematopoietic cells in the induction of immune dysregulation**

The strong MLR against autologous clonal CD34+ progenitors observed in patients with RAEB was inversely correlated with marrow cellularity. All four patients with a strong APLR had marrow cellularity of <35% and marrow blast cell percentage of 5–10% (RAEB-1). These patients, although severely pancytopenic and transfusion dependent, had a delayed evolution to AML or they did not progressed at all. In two of them who progressed, marrow cellularity was increased and in a new APLR, performed at the time of AML progression, lymphocyte activation against autologous blasts had been abrogated [86]. Thus, it appears that lymphocyte activation in patients with RAEB is rather inversely correlated with leukemic burden, but immunologic memory for this reaction is maintained and patients who lose their immune activation during leukemic transformation may regain it following the achievement of remission. Moreover, maintenance of remission is completely dependent on the presence of autologous cytotoxicity against leukemic cells, mainly exerted by NK cells. This cytotoxic reaction is disappeared upon relapse of the leukemia [87]. Unfortunately, immune activation against clonal cells is not the rule and T cells in MDS (particularly Tγδ cells) may respond poorly, if not at all, even following IL-2 stimulation, despite normal IL-2R expression, demonstrating impaired immune surveillance against the clone [51]. Indeed, the Czech group did not find any significant lymphocyte activation in eight out of nine patients tested and confirmed the nonclonal origin of the T cells [71].

Clonal dendritic cells can also induce T-cell stimulation in AML. Proliferative reaction against these cells is high but results in the generation of cytotoxic T lymphocytes with low activity against autologous or allogeneic nonleukemic targets [88]. Dendritic cells of MDS patients in Allo-MLR systems are poor stimulators for both normal and MDS-derived T cells, indicating an impaired antigen presenting capacity. These cells, generated from CD34+ cells, although immunophenotypically normal, were significantly decreased as were also the populations or circulating myeloid- and plasmacytoid-derived dendritic cells, confirming ineffective "den‐ dritopoiesis" [89] and produce less IL-12 and more IL-10 in response to LPS and IFN-γ showing qualitative and quantitative defects of cytokine production.

Blast cells exert direct suppressor activity on the activation, TH1 polarization and proliferation of T lymphocytes. This activity is mediated through protein substances transcribed via the NF-AT and NF-kB signals by inhibition of transition from G0 to G1 phase [90]. Upon NK cells, leukemic cells induce impaired killing capacity, reduced TNF-α and IFN-γ production, reduced CD107α degranulation, downregulation of the NKp46- and upregulation of the NKG2A receptor expression, effects directed via IL-10, and favoring clonal escape and expansion [91]. In other instances, however, blast cells induce lymphocyte activation, as previously described, with the production of IL-2, IL-4, IL-10, IL-13, and IFN-γ. Lymphocyte culture supernatants, when further activated with IL-2, generate strong cytotoxic LAK and NK cells inducing lysis of autologous and allogeneic leukemic cells [92]. In the majority of cases, immune effector function of NK-T and NK cells, observed in some patients with MDS, are abolished on leukemic transformation.

#### **7. Immunopathogenetic aspects of myelodysplastic syndromes**

From the pathogenetic point of view it appears that an initial, harmful event (viral, drug, irradiation, etc.), affecting the pluripotent hematopoietic stem cell compartment in the bone marrow, may antigenically alter a minor population of these cells. Even when the consequen‐ ces of the harmful event are negligible and maturation and differentiation processes might remain almost intact, it is possible that an immunologic reaction could be initiated. This reaction is directed against the even minimally modulated hematopoietic progenitor cell population. Indeed, the strength of the autologous cytotoxic immune reaction, frequently accompanying the emergence of a dysplastic clone, is not related to the complexity/severity of the cytogenetic abnormalities, and a minor genetic damage may induce a strong reaction. Conversely, complex chromosomal aberrations and other gross genetic damage, leading to hematopoietic failure, may induce a weak or not any immune reaction. This reaction may be less specific and may also generate cytotoxicity, not only against the affected cells, but also to the unaffected/normal hematopoietic progenitors, inducing apoptosis and resulting in stemcell depletion and hematopoietic failure. Soluble factors (cytokines) released by the activated lymphocytes might also harm accessory/stromal cells. This cascade of events usually leads to aplastic anemia. When the initial harmful event induces deeper genetic damage in a pluripo‐ tent stem cell and this cell, although genetically altered, succeeds in escaping from apoptotic cell death may generate an abnormal (dysplastic) clone. Clonal cells continue to trigger the immunocompetent cells, but the latter although activated cannot eliminate the clonal cells, which continue to escape, gradually expand, and suppress the unaffected/normal stem cell compartment through at least two mechanisms:

activation in patients with RAEB is rather inversely correlated with leukemic burden, but immunologic memory for this reaction is maintained and patients who lose their immune activation during leukemic transformation may regain it following the achievement of remission. Moreover, maintenance of remission is completely dependent on the presence of autologous cytotoxicity against leukemic cells, mainly exerted by NK cells. This cytotoxic reaction is disappeared upon relapse of the leukemia [87]. Unfortunately, immune activation against clonal cells is not the rule and T cells in MDS (particularly Tγδ cells) may respond poorly, if not at all, even following IL-2 stimulation, despite normal IL-2R expression, demonstrating impaired immune surveillance against the clone [51]. Indeed, the Czech group did not find any significant lymphocyte activation in eight out of nine patients tested and confirmed the

Clonal dendritic cells can also induce T-cell stimulation in AML. Proliferative reaction against these cells is high but results in the generation of cytotoxic T lymphocytes with low activity against autologous or allogeneic nonleukemic targets [88]. Dendritic cells of MDS patients in Allo-MLR systems are poor stimulators for both normal and MDS-derived T cells, indicating an impaired antigen presenting capacity. These cells, generated from CD34+ cells, although immunophenotypically normal, were significantly decreased as were also the populations or circulating myeloid- and plasmacytoid-derived dendritic cells, confirming ineffective "den‐ dritopoiesis" [89] and produce less IL-12 and more IL-10 in response to LPS and IFN-γ showing

Blast cells exert direct suppressor activity on the activation, TH1 polarization and proliferation of T lymphocytes. This activity is mediated through protein substances transcribed via the NF-AT and NF-kB signals by inhibition of transition from G0 to G1 phase [90]. Upon NK cells, leukemic cells induce impaired killing capacity, reduced TNF-α and IFN-γ production, reduced CD107α degranulation, downregulation of the NKp46- and upregulation of the NKG2A receptor expression, effects directed via IL-10, and favoring clonal escape and expansion [91]. In other instances, however, blast cells induce lymphocyte activation, as previously described, with the production of IL-2, IL-4, IL-10, IL-13, and IFN-γ. Lymphocyte culture supernatants, when further activated with IL-2, generate strong cytotoxic LAK and NK cells inducing lysis of autologous and allogeneic leukemic cells [92]. In the majority of cases, immune effector function of NK-T and NK cells, observed in some patients with MDS, are

**7. Immunopathogenetic aspects of myelodysplastic syndromes**

From the pathogenetic point of view it appears that an initial, harmful event (viral, drug, irradiation, etc.), affecting the pluripotent hematopoietic stem cell compartment in the bone marrow, may antigenically alter a minor population of these cells. Even when the consequen‐ ces of the harmful event are negligible and maturation and differentiation processes might remain almost intact, it is possible that an immunologic reaction could be initiated. This reaction is directed against the even minimally modulated hematopoietic progenitor cell

nonclonal origin of the T cells [71].

30 Myelodysplastic Syndromes

abolished on leukemic transformation.

qualitative and quantitative defects of cytokine production.

(1) Immune effector cytotoxic cells can destroy more easily the nonclonal/normal progenitor cells as a result of clonal cell escape from the immune attack.

(2) Secondary genetic alterations occurring gradually provide growth advantage to the clonal cells.

Thus, immune activation may perpetuate and when cytotoxic activity is ineffective and incapable to eliminate clonal cells, it becomes an "immunologic abnormality." The more effective the immune activation, the higher the degree of apoptosis induced, affecting more and more marrow cellularity and creating a syndrome mostly similar to aplastic anemia. Therefore, the decreased marrow cellularity observed in some marrow failure syndromes might be considered an "adverse event" of an effective immune reaction capable to restrict the growth of the abnormal/mutated/genetically altered clonal cell population. On rare occasions, the intensive immune activation, augmented by an infection or a blood transfusion, may be capable to completely eradicate the dysplastic clone leading to spontaneous complete remis‐ sion even after evolution to AML. In contrast, when immune activation is ineffective, clonal expansion and evolution continues unimpededly until the stage of AML. At this stage, either passively, due to high "antigenic burden," or actively, through mechanisms, induced by the leukemic cells, immune tolerance or immune paralysis is established abrogating further immune reaction [93]. In rare instances, even after evolution, immune activation may be maintained and result in an oligoblastic/hypoplastic AML. Conversely, when immune activation is abolished early or when the dysplastic/neoplastic clone achieves in earning immune tolerance, the evolution might be uneventful and lead to a hypercellular AML.

About 10–15% of MDS patients at initial presentation have a hypoplastic marrow (cellularity ≤30%). These patients exhibit more severe cytopenias, various degrees of trilineage dysplasia, more prominent immune abnormalities, and usually a normal karyotype or single chromoso‐ mal abnormalities. Although hypoplastic MDS share many similarities with aplastic anemia, different molecular mechanisms of marrow damage have been identified between them and other/nonhypoplastic MDS. Among them development of oligoclonal expansion of cytotoxic T lymphocytes, overexpression of TRAIL- and Fas ligand-induced apoptosis, underexpression of Flice-like inhibitory protein long isoform (FLIPL), and increased production of IFN-γ and TNF-α are included. Patients with hypolastic MDS have more stable clinical course and lower evolution rates in relation to patients with nonhypoplastic disease of the same FAB/WHO categories. They show good response to treatment with corticosteroids, cyclosporine-A, antithymocyte globulin, or alemtuzumab, and to various combinations of the above. Overall survival varies and if patients will not succumb to a severe infection, they may retain a prolonged leukemia-free survival [94, 95].

Suppressor/cytotoxic autoimmune reactions are more frequently identified among lower risk MDS, have specificity against the pluripotent or an early committed, usually erythropoietic progenitor cell, and are associated with higher degree of marrow apoptosis. In the majority of cases, the autoimmune process includes the production of specific antierythroblastic antibod‐ ies without the positivity of direct antiglobulin test. The production of such IgG autoantibodies can be provoked *ex vivo* following antigenic stimulation [96]. These patients show higher caspace-3 activity and lower TNF-α and IL-4 production. Analysis of the total IgM and IgG antibody repertoire in 10 MDS patients without prominent autoimmune disease or known autoantibody and in 10 healthy controls revealed different patterns of antibodies against selfantigens in MDS patients from those of controls, and patterns of IgG antibodies had distinct profiles implying disturbed self-recognition related to pathogenetic mechanisms of the disease [97].

Increased marrow apoptosis is a dominant feature of MDS and affects all hematopoietic cell compartments from the more immature-undifferentiated to the mature and recognizable cells. The apoptotic rate of CD34+ cells in normal subjects has been calculated at about 1%, whereas in MDS, it ranges from 3 to 15%. Higher apoptotic rate is usually found in patients with early MDS and in few patients with an excess of blasts [98]. Apoptotic rate may vary in the same patient at different time points reflecting also the evolution of mutational status of the clonal cells. Specific cytogenetic abnormalities, such as trisomy 8 have been associated with higher degree of apoptosis. Apoptosis is a multifactorial process in MDS with a possible contribution of the immune effector cells. Clonal cells' death could create abnormal structures with potentially (auto)antigenic properties and apoptosis can represent the causative factor of the initiation of autologous cytotoxic immune reaction. Indeed, increased apoptotic cell rate has been associated with higher marrow cytotoxic T-cell infiltration and in many instances by oligoclonal T cells in MDS patients, who also express the TIA-1 antigen on their hematopoietic cells [99]. The proapoptotic marrow microenvironment triggers stromal cells to produce IL-32, which in turn induces further TNF-α transcription, thus establishing a vicious cycle. IL-32 expression has been found many folds higher in the stromal cells of MDS patients, rendering this cytokine a specific stromal cell marker for MDS [100].

Although immune activation plays the dominant pathogenetic role for the generation of marrow failure in aplastic anemia, in MDS this cannot be easily identified in every individual patient. In other words, it is not identifiable which part of the hematopoietic failure results from the clonal disorder *per se* and which is attributed to the immune activation. This fact could explain, at least in part, why there is not a uniform response rate to the immunosuppressive treatment and this rate may vary widely in different series of patients, irrespective of FAB or WHO category, cytogenetics, and the severity of the cytopenias [101].

other/nonhypoplastic MDS. Among them development of oligoclonal expansion of cytotoxic T lymphocytes, overexpression of TRAIL- and Fas ligand-induced apoptosis, underexpression of Flice-like inhibitory protein long isoform (FLIPL), and increased production of IFN-γ and TNF-α are included. Patients with hypolastic MDS have more stable clinical course and lower evolution rates in relation to patients with nonhypoplastic disease of the same FAB/WHO categories. They show good response to treatment with corticosteroids, cyclosporine-A, antithymocyte globulin, or alemtuzumab, and to various combinations of the above. Overall survival varies and if patients will not succumb to a severe infection, they may retain a

Suppressor/cytotoxic autoimmune reactions are more frequently identified among lower risk MDS, have specificity against the pluripotent or an early committed, usually erythropoietic progenitor cell, and are associated with higher degree of marrow apoptosis. In the majority of cases, the autoimmune process includes the production of specific antierythroblastic antibod‐ ies without the positivity of direct antiglobulin test. The production of such IgG autoantibodies can be provoked *ex vivo* following antigenic stimulation [96]. These patients show higher caspace-3 activity and lower TNF-α and IL-4 production. Analysis of the total IgM and IgG antibody repertoire in 10 MDS patients without prominent autoimmune disease or known autoantibody and in 10 healthy controls revealed different patterns of antibodies against selfantigens in MDS patients from those of controls, and patterns of IgG antibodies had distinct profiles implying disturbed self-recognition related to pathogenetic mechanisms of the disease

Increased marrow apoptosis is a dominant feature of MDS and affects all hematopoietic cell compartments from the more immature-undifferentiated to the mature and recognizable cells. The apoptotic rate of CD34+ cells in normal subjects has been calculated at about 1%, whereas in MDS, it ranges from 3 to 15%. Higher apoptotic rate is usually found in patients with early MDS and in few patients with an excess of blasts [98]. Apoptotic rate may vary in the same patient at different time points reflecting also the evolution of mutational status of the clonal cells. Specific cytogenetic abnormalities, such as trisomy 8 have been associated with higher degree of apoptosis. Apoptosis is a multifactorial process in MDS with a possible contribution of the immune effector cells. Clonal cells' death could create abnormal structures with potentially (auto)antigenic properties and apoptosis can represent the causative factor of the initiation of autologous cytotoxic immune reaction. Indeed, increased apoptotic cell rate has been associated with higher marrow cytotoxic T-cell infiltration and in many instances by oligoclonal T cells in MDS patients, who also express the TIA-1 antigen on their hematopoietic cells [99]. The proapoptotic marrow microenvironment triggers stromal cells to produce IL-32, which in turn induces further TNF-α transcription, thus establishing a vicious cycle. IL-32 expression has been found many folds higher in the stromal cells of MDS patients, rendering

Although immune activation plays the dominant pathogenetic role for the generation of marrow failure in aplastic anemia, in MDS this cannot be easily identified in every individual patient. In other words, it is not identifiable which part of the hematopoietic failure results from the clonal disorder *per se* and which is attributed to the immune activation. This fact could

prolonged leukemia-free survival [94, 95].

32 Myelodysplastic Syndromes

this cytokine a specific stromal cell marker for MDS [100].

[97].

### **8. Immunosuppressive/immunomodulating treatment applied to patients with MDS**

Corticosteroids are the most widely used immunosuppressive treatment administered to patients with MDS and autoimmune diseases [17, 20]. Response rates vary broadly and the required dose depends on the type of autoimmune disease, MDS subtype, chronicity of the condition, and other factors. Although symptom resolution may be fast, autoimmune disease may relapse during tapering, demanding higher doses, which may not be tolerable by elderly patients. Thus, corticosteroids usually lead to partial or transient response and second-line treatment with other agents is necessary. Steroids may also benefit hematopoiesis, improving cytopenias and reducing RBC transfusion needs. Responses are mainly seen in patients with lower risk IPSS, with a specific profile, but also by some patients with RAEB [102] and may be long-lasting and maintained with small maintenance steroid doses.

Cyclosporin-A (Cy-A) is the second more widely used immunosuppressive agent and has also been used in combination with cytotoxic chemotherapy as a modulator of multidrug resist‐ ance, which is commonly found in higher risk MDS and in AML following MDS. Cy-A is effective even at lower doses, aiming to achieve serum levels lower than those desirable in aplastic anemia and in allogeneic stem cell transplantation and therefore is well-tolerated and induces durable remissions [103, 104]. Retrospective evaluation of 50 patients showed a hematological improvement and particularly an erythroid response in 60%. Better response was achieved by patients with hypoplastic marrow, favorable karyotype, or carrying the DRB1\*1501 allele [105]. The NIH group has reported more frequent expression of the HLA-DR2/HLA-DR15 allele in patients with MDS and aplastic anemia compared to a control population and an association of the expression of this allele with a favorable response to immunosuppression [106]. Cy-A added to T-lymphocyte cultures decreases IFN-γ- but not Fas-L production and lead to abrogation of the inhibitory activity of the supernatant on hematopoietic colony formation. However, the growth of secondary colonies continues to be decreased due to low number of pluripotent CD34+ progenitors.

Probably the most effective immunosuppressive treatment is antithymocyte globulin (ATG), which has been given to MDS patients with any marrow cellularity [101, 104]. Hematopoietic improvement is achieved following elimination of the autoreactive cytotoxic T cells and may result in restoration of the dysplastic marrow and peripheral blood morphology. In many instances cytogenetic complete remission has also been reported, whereas in others, hemato‐ logic remission is not accompanied by cytogenetic remission. In these cases, most probably immune activation mainly suppresses nonclonal hematopoiesis without significantly disturb‐ ing the dysplastic clone. Finally, rapid evolution to AML or increase of marrow blasts despite hematological improvement has occasionally been reported following treatment with Cy-A or ATG [107]. In these cases, immune activation may effectively suppress clonal cells and its abrogation has favored the unimpeded clonal expansion and evolution. Mofetil mycopheno‐ late (MMF) or alemtuzumab can be used when corticosteroids and/or Cy-A are ineffective or contraindicated, or when severe adverse effects emerge, but the experience with these agents is limited. The main drawback of immunosuppression is that combined with the usually coexisting neutropenia substantially increases the risk for common and opportunistic infec‐ tions, even when all prophylactic measures are applied. Cy-A, in particular, may further impair previously existed renal failure and may induce various adverse events as a result of phar‐ macodynamic interactions to patients concommittantly treated with many other drugs.

Immune-stimulating treatment, targeting NK-/NK-T cells and aiming to generate cytotoxic Tcell activity and eliminate the dysplastic clone, has been associated with rather disappointing results. A promising message is that newer immunomodulating drugs, such as lenalidomide, appear to increase NK T cells and improve their function, including cytokine production, although this is not the major mechanism of action of the drug [108]. Hypomethylating agents, currently used particularly in higher risk patients, when effective and leading to complete response may also benefit autoimmune or hyperimmune clinical syndromes associated with MDS. It has also been suggested that 5-azacytidine has an independent immune-modulating activity and that remissions of the auto-/hyperimmune syndrome may occur independently of the induction of hematological and cytogenetic response, and might also be effective in cases in which other immunosuppressive treatments have been proved ineffective [109].

The injudicious use of immunosuppressive treatment in MDS may become a trench knife [110]. Patients exhibiting overactive immune response, but clonal hematopoiesis, even when sharing a hypoplastic bone marrow, might need even more effective immune activation to wear down the dysplastic clone. Similarly, patients with an established clonal disease, but without any immune activation, could potentially gain benefit with the administration of immune stimu‐ lation in an effort to eliminate the clone. On the other hand, abrogation of an overactive immune stimulation should be attempted when this activation suppresses primarily the residual normal/nonclonal hematopoiesis and minimally disturbs the development of the abnormal/dysplastic clone. When immune activation/reaction status cannot be identified and/ or quantified, in the middle of established dysplastic hematopoiesis, a course of moderately strong immunosuppressive treatment with corticosteroids and/or cyclosporine could be administered, and in cases of a favorable response, careful tapering of the drugs should be tested in an effort to maintain the obtained response.

#### **Author details**

Argiris Symeonidis\* and Alexandra Kouraklis-Symeonidis

\*Address all correspondence to: argiris.symeonidis@yahoo.gr

Hematology Division, Department of Internal Medicine, University Hospital of Patras, Patras, Greece

#### **References**

ATG [107]. In these cases, immune activation may effectively suppress clonal cells and its abrogation has favored the unimpeded clonal expansion and evolution. Mofetil mycopheno‐ late (MMF) or alemtuzumab can be used when corticosteroids and/or Cy-A are ineffective or contraindicated, or when severe adverse effects emerge, but the experience with these agents is limited. The main drawback of immunosuppression is that combined with the usually coexisting neutropenia substantially increases the risk for common and opportunistic infec‐ tions, even when all prophylactic measures are applied. Cy-A, in particular, may further impair previously existed renal failure and may induce various adverse events as a result of phar‐ macodynamic interactions to patients concommittantly treated with many other drugs.

Immune-stimulating treatment, targeting NK-/NK-T cells and aiming to generate cytotoxic Tcell activity and eliminate the dysplastic clone, has been associated with rather disappointing results. A promising message is that newer immunomodulating drugs, such as lenalidomide, appear to increase NK T cells and improve their function, including cytokine production, although this is not the major mechanism of action of the drug [108]. Hypomethylating agents, currently used particularly in higher risk patients, when effective and leading to complete response may also benefit autoimmune or hyperimmune clinical syndromes associated with MDS. It has also been suggested that 5-azacytidine has an independent immune-modulating activity and that remissions of the auto-/hyperimmune syndrome may occur independently of the induction of hematological and cytogenetic response, and might also be effective in cases

in which other immunosuppressive treatments have been proved ineffective [109].

and Alexandra Kouraklis-Symeonidis

Hematology Division, Department of Internal Medicine, University Hospital of Patras, Patras,

tested in an effort to maintain the obtained response.

\*Address all correspondence to: argiris.symeonidis@yahoo.gr

**Author details**

34 Myelodysplastic Syndromes

Argiris Symeonidis\*

Greece

The injudicious use of immunosuppressive treatment in MDS may become a trench knife [110]. Patients exhibiting overactive immune response, but clonal hematopoiesis, even when sharing a hypoplastic bone marrow, might need even more effective immune activation to wear down the dysplastic clone. Similarly, patients with an established clonal disease, but without any immune activation, could potentially gain benefit with the administration of immune stimu‐ lation in an effort to eliminate the clone. On the other hand, abrogation of an overactive immune stimulation should be attempted when this activation suppresses primarily the residual normal/nonclonal hematopoiesis and minimally disturbs the development of the abnormal/dysplastic clone. When immune activation/reaction status cannot be identified and/ or quantified, in the middle of established dysplastic hematopoiesis, a course of moderately strong immunosuppressive treatment with corticosteroids and/or cyclosporine could be administered, and in cases of a favorable response, careful tapering of the drugs should be


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## **Disorders Mimicking Myelodysplastic Syndrome and Difficulties in its Diagnosis**

Lale Olcay and Sevgi Yetgin

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64422

#### **Abstract**

Myelodysplastic morphology of blood cells can be encountered not only in myelodys‐ plastic syndrome (MDS) but also in nonclonal disorders like viral, bacterial, parasitic infections, juvenile rheumatoid arthritis, polyarteritis nodosa, immune thrombocyto‐ penic purpura (ITP), iron deficiency anemia, megaloblastic anemia, dysgranulopoietic neutropenia, congenital neutropenia, cases with microdeletion 22q11.2, malignant lymphoma, after administration of granulocyte colony stimulating factor, chemothera‐ py, steroids, smoking, alcohol, posttransplantation, copper deficiency also, together with or without cytopenia. Absence of cytogenetic abnormality in 50–70% of cases with MDS, some overlapping morphological and/or pathophysiological features make it challeng‐ ing to differentiate between MDS and other diseases/disorders like aplastic anemia, refractory ITP, copper deficiency. Transient genetic abnormalities including monoso‐ my 7 in megaloblastic anemia; increased immature myeloid cells in bone marrow of cases with copper, vitamin B12, or folic acid deficiency in the setting of cytopenia and dysmorphism may also lead to the misdiagnosis of MDS. On the other hand, there are also cases of transient MDS. In this chapter, a literature is be presented to draw atten‐ tion of the readers on the disorders that mimic MDS. Additionally, our personal experiences are also be shared. Awareness of disorders mimicking MDS may prevent over- or underdiagnosis of MDS.

**Keywords:** secondary myelodysplasia, cell death, cell cycle, transient MDS, apoptosis, rapid cell senescence

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **1. Introduction**

Myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis in bone marrow and cytopenias in peripheral blood. It is heterogeneous reflected by a number of subgroups with different characteristics both in adulthood and childhood [1–11].

A single diagnostic parameter specific to MDS has not been discovered yet, and a considerable number of patients with MDS lack chromosomal abnormality [1, 4]. Currently, the diagnosis of MDS is mainly dependent on quantitative and qualitative dysplastic abnormalities [5]. Establishment of special characteristics of dysplasia like the number of dysplastic cell lines, the percentage of the dysplastic cells, and characteristic megakaryocytes as in del(5q) syndrome are critical in order to be able to assess which subgroup of MDS the patient fits according to the World Health Organization (WHO) classification. Because uni- or multilineage dysplasia may be the only criterion that differentiates the subgroups refractory cytopenia with unilineage dysplasia (RCUD) and refractory cytopenia with multilineage dysplasia (RCMD) [1, 5, 6, 10, 11]. Additionally, for the lineage to be considered as dysplastic, at least 10% of the lineage should display displastic findings [1, 2, 5, 6, 8, 10, 11] both in adulthood and childhood MDS.


**Table 1.** Recent definitions of morphological features of myelodysplasia (adulthood) [2, 5].

Nearly half of which is constituted by refractory cytopenia of childhood (RCC) [4]. Minimal diagnostic criteria for childhood MDS require fulfillment of at least two of the following criteria: sustained, unexplained cytopenia (neutropenia, thrombocytopenia, or anemia); at least bilineage morphologic myelodysplasia; acquired clonal cytogenetic abnormality in hematopietic cells; increased blast count (>5%) [3].

**1. Introduction**

46 Myelodysplastic Syndromes

adulthood and childhood MDS.



Incomplete -Hemoglobinization -Fringed cytoplasm -Cytoplasmic bridging

Irregular nuclear edges Internuclear bridging -Karyorrhexis -Multinuclearity -Nuclear hyperlobulation



Peripheric blood -Anisocytosis

Bone marrow -Nuclear budding

Myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis in bone marrow and cytopenias in peripheral blood. It is heterogeneous reflected by a number of subgroups with different characteristics both in adulthood and childhood [1–11].

A single diagnostic parameter specific to MDS has not been discovered yet, and a considerable number of patients with MDS lack chromosomal abnormality [1, 4]. Currently, the diagnosis of MDS is mainly dependent on quantitative and qualitative dysplastic abnormalities [5]. Establishment of special characteristics of dysplasia like the number of dysplastic cell lines, the percentage of the dysplastic cells, and characteristic megakaryocytes as in del(5q) syndrome are critical in order to be able to assess which subgroup of MDS the patient fits according to the World Health Organization (WHO) classification. Because uni- or multilineage dysplasia may be the only criterion that differentiates the subgroups refractory cytopenia with unilineage dysplasia (RCUD) and refractory cytopenia with multilineage dysplasia (RCMD) [1, 5, 6, 10, 11]. Additionally, for the lineage to be considered as dysplastic, at least 10% of the lineage should display displastic findings [1, 2, 5, 6, 8, 10, 11] both in

**Dyserythropoiesis Dysgranulopoiesis Dysmegakaryopoiesis**










**Table 1.** Recent definitions of morphological features of myelodysplasia (adulthood) [2, 5].


1 Megaloblastic changes: At least 1.5 times the size of a normal poly- or orthochromatic erythroblast with coarse condensation of chromatin and an increased nuclear-to-cytoplasmic ratio or orthochromatic erythroblasts with decreased nuclear-to-cytoplasmic ratio and at least double the size of a normal erythrocyte of the same maturational state.

2 Cytoplasmic granules or inclusions: Presence of granules or nuclear fragments that can be definitely differentiated from ribosomal RNA.

3 Bizarre nuclear shape: Abnormal nuclear shape, including irregularly lobulated nuclei of segmented granocytes with chromatin clumping or large twisted bands, large bands or metamyelocytes, multinuclearity (two distinctly separated neutrophilic bands or segmented nuclei).

4 A- or hypogranularity: Neutrophilic or azurophilic granules should be markedly or completely absent and the cytoplasm of mature neutrophilic granulocytes has to stain pale blue/gray or translucent in the Romanowsky-Giemza stain. All maturation stages except blast cells should be affected.

5 Nuclear/cytoplasmic (N/C) asyncrony: Mature neutrophilic granulocytes and metamyelocytes with basophilic cytoplasm and myelocytes with neutrophilic cytoplasm.

6 Pseudo-Pelger anomaly: Mature granulocytes with either a centrally located round to ovoid nucleus (monolobated type) or two round nuclei of similar size connected by a slender chromatin bridge (bilobated type).

7 Micromegakaryocytes: Mononucleated megakaryocyte with a size comparable to that of a promyelocyte or less, lacking features of a blast cell.

8 Small binucleated megakaryocyte: Small megakaryocyte with the size of a micromegakaryocyte or slightly larger, with two round well-separated nuclei.

9 Megakaryocyte with small round separated nuclei: Megakaryocytes of any size with multiple, at least three, round separated nuclei.

10Megakaryocytes with nonlobated round nucleus: Megakaryocytes of normal or reduced size with a nonlobated round nucleus and a mature granular cytoplasm.

**Table 2.** Morphological features of myelodysplasia (childhood-EWOG-MDS Group, 2005) [13].

The unilineage dysplasia in RCUD of adult MDS should have lasted for at least 6 months if no clonal cytogenetic abnormality is found and/or ring sideroblasts are less than 15% [12], so for these patients a repeated bone marrow examination is recommended after a 6 months' observation [5].

The dysplastic changes have been standardized in childhood [13, 14] and adulthood MDS [2, 5, 15, 16] (**Tables 1**–**4**) being restricted to three lineages as erythroid, granulocytic, and megakaryocytic lineages. Although monocytes are rarely affected in MDS, their presence rather is associated with CMML and AML [5].


**Table 3.** Morphological features of myelodysplasia in refractory cytopenia of childhood (RCC) (WHO, 2008) [14].


**Table 4.** Other parameters for myelodysplasia that were suggested previously but had not been included in the recent guidelines [15, 16].

On the other hand, myelodysplastic findings in blood cells arise due to any challenge during the course of normal differentiation and therefore these changes can be encountered not only in clonal disorders like MDS (primary myelodysplasia), but also in various nonclonal disorders affecting bone marrow like viral, bacterial, parasitic infections [17–27], autoimmune disorders (juvenile rheumatoid arthritis, polyarteritis nodosa, systemic lupus erythematosis, immune thrombocytopenic purpura) [17, 28–32], hemophagocytic histiocytosis (HLH), nutritional problems (malnutrition, iron deficiency anemia, megaloblastic anemia, copper deficiency, vitamin D deficiency, hyper vitaminosis A) [27, 33–43], neutropenia (congenital dysgranulo‐ poietic, congenital severe, idiopathic) [32, 44, 45], inherited disorders [27, 46, 47], malign lymphoma [48], due to effects of drugs and toxins [17, 27, 49–54], during posttransplantation period [27, 55], and other reasons [27] also and called "secondary myelodysplasia" [27]. These findings are neutrophils with nuclear shape, hypoagranulation, abnormal nuclei, cytoplasm and granulation, anisocytosis, poikilocytosis, microspherocytes, giant thrombocytes, lympho‐ cytes with cytoplasmic protrusions and vacuoles, monocytes with dysmorphic nuclei, cytoplasmic vacuoles and cytoplasmic protrusions, chromatin clumping, nucleocytoplasmic asyncrony, interchromatin bridges between erythroid precursors, oligonuclear megakaryo‐ cytes, naked megakaryocyte nuclei and cytoplasm, most of which have been included in the dysplasia criteria in MDS of childhood [13, 14], and adulthood [2, 15, 16].

While MDS is potentially preleukemic, disorders with secondary myelodysplasia are not neoplastic or preleukemic and are reversible when the underlying factor is removed [27].

Such cases with additional cytopenia in one or more cell lines, due to transient suppression of hematopoiesis may erroneously lead the physician to the diagnosis of MDS, especially when no cytogenetic abnormality can be attained. Additionally, assessment of morphological abnormalities in MDS is still not completely objective [56], in spite of that a number of dysmorphic findings were simplified, categorized [2, 13–16] and cut-off values were estab‐ lished [5]. This situation is valid especially for low-risk MDS cases without excess blasts and any detectable cytogenetic abnormalities. Additionally, necessity to wait without definite diagnosis and therefore therapy for at least 6 months in cytopenia cases with unilineage dysplasia [5, 12] is distressing for the patient and the family.

On the other hand, it is also challenging to differentiate cases which present as ordinary aplastic anemia, refractory immune thrombocytopenic purpura (ITP) [31], chronic neutropenia [44, 45] from MDS [4, 9, 14, 57–60]. Transient MDS or MDS-like disorders with or without [61–70] chromosomal abnormalities, acute myeloblastic leukemia (AML) cases with low blast cell count [71] should also be considered for accurate diagnosis. Additionally, it should not be forgotten that autoimmune disorders may also be a component of MDS itself [72–74] and there may be cases complying the criteria of other MDS subtypes like idiopathic cytopenia of undetermined significance (ICUS), idiopathic dysplasia of undetermined significance (IDUS) [5, 7, 58].

This chapter reviews on these diagnostic problems, in the following order:


The dysplastic changes have been standardized in childhood [13, 14] and adulthood MDS [2, 5, 15, 16] (**Tables 1**–**4**) being restricted to three lineages as erythroid, granulocytic, and megakaryocytic lineages. Although monocytes are rarely affected in MDS, their presence

> -Megakaryocytes with variable size and separated nuclei or round nuclei (absence of megakaryocytes

> > -Megakaryocyte fragments -Giant granules in thrombocyte -Bothryoid nucleus in megakaryocytes -Hypogranulation in megakaryocytes

does not rule out RCC)

**Dyserythropoiesis Dysgranulopoiesis Dysmegakaryopoiesis**

**Table 3.** Morphological features of myelodysplasia in refractory cytopenia of childhood (RCC) (WHO, 2008) [14].

**Dyserythropoiesis Dysgranulopoiesis Dysmegakaryopoiesis**




**Table 4.** Other parameters for myelodysplasia that were suggested previously but had not been included in the recent

On the other hand, myelodysplastic findings in blood cells arise due to any challenge during the course of normal differentiation and therefore these changes can be encountered not only in clonal disorders like MDS (primary myelodysplasia), but also in various nonclonal disorders affecting bone marrow like viral, bacterial, parasitic infections [17–27], autoimmune disorders (juvenile rheumatoid arthritis, polyarteritis nodosa, systemic lupus erythematosis, immune thrombocytopenic purpura) [17, 28–32], hemophagocytic histiocytosis (HLH), nutritional problems (malnutrition, iron deficiency anemia, megaloblastic anemia, copper deficiency, vitamin D deficiency, hyper vitaminosis A) [27, 33–43], neutropenia (congenital dysgranulo‐ poietic, congenital severe, idiopathic) [32, 44, 45], inherited disorders [27, 46, 47], malign lymphoma [48], due to effects of drugs and toxins [17, 27, 49–54], during posttransplantation period [27, 55], and other reasons [27] also and called "secondary myelodysplasia" [27]. These




not be complied)

Neutrophilic lineage



rather is associated with CMML and AML [5].

Abnormality -Abnormal nuclear lobulation -Multinuclear cells -Nuclear bridges

48 Myelodysplastic Syndromes


guidelines [15, 16].




#### **2. Nonclonal disorders which present as dysplasia and cytopenia**

#### **2.1. Viral and bacterial infections**

In a pilot and unpublished study that we carried on in our clinic, we compared the dysmorphic parameters in the neutrophils of patients with viral (n:6; infections: rubella, rubeola, viral eruption of unknown origin, Ebstain Barr virus infection), bacterial (n:7; infections: preseptal cellulitis, urinary infection, tonsillitis, maxillary sinusitis, lymphadenitis, otitis media) infections and those of healthy controls. We found that the neutrophil diameter of those with bacterial infections; the percentage of pseudo Pelger-Huet cells and irregular distribution of granules in both viral and bacterial infections; the percentage of chromatin clumping in viral diseases were higher than the control. These findings showed that nonspecific infections can also give rise to dysmorphic findings in neutrophils.

In another study, we reported that those with bacterial diseases additionally displayed comparable diameter, macropolycyte (neutrophils with diameter >14 μm) percentage, bizarre nucleus, irregular distribution of granules with those of pretreatment ITP who also displayed myelodysplasia [17].

Striking dyserytropoiesis was reported in tuberculosis [27]. Several viral infections which closely mimick MDS will be delineated below.

#### *2.1.1. Parvovirus infection*

In the literature, there are cases of parvovirus infection, with [19–21] or without [22] immu‐ nodeficiency or chronic hemolytic anemia which transiently or chronically mimicked MDS or dyserythropoietic anemia [22]. Among them the two [19, 20] are of note.

The reported case of Hasle et al. [19] was an 8-year-old, previously healthy boy who admitted to the hospital with severe anemia, moderate thrombocytopenia, and granulocytopenia and a 2 weeks' history of intermittent fever. Physical examination was normal except for pallor. Bone marrow was hypercellular with marked erythroblastopenia and maturation arrest of the erythropoietic cell line. No giant pronormoblast and hemophagocytosis was noted. Dysplasia in myeloid and megakaryocytic lineage was evident. He had increased immunoglobulin (Ig) M, low IgG, slightly decreased natural killer (NK) cells which reduced during follow-up; impaired in vitro proliferation of blood mononuclear cells on stimulation.

The patient was assumed as MDS and was administered prednisolon, androgenic steroid, cyclophosphamid and cyclosporine, IgG infusion and frequent blood transfusions and developed hemochromatosis and hepatosplenomegaly. Thrombocytopenia deepened; hemoglobin transiently normalized. Parvovirus antibody studies revealed negative but when polymerase chain reaction technique became available, serum samples of the previous two years of the disease course and bone marrow smears were found positive for parvovirus infection.

The reported case of Baurmann et al. [20] was a 36-year-old, previously healthy woman who admitted to the hospital with fever, pancytopenia, and atypical lymphoid cells with dysplastic hematopoietic changes. She had frank splenomegaly, slightly increased bilirubin, lactate dehydrogenase (LDH), negative Coombs test. Since the bone marrow was hypocellular with multiple abnormal megakaryocytes, absence of erythropoiesis and 15% blasts carrying monocytic and histiocytic characteristics, she was diagnosed as MDS-refractory anemia with excess blasts (RAEB). A second bone marrow aspiration performed 6 days after admission revealed hypercellularity, no excess of blasts, erythropoietic hyperplasia with giant proery‐ throblasts, megakaryocytes which were in normal number but still dysplastic.

Parvovirus antibodies and DNA were positive while the serologic tests for other viruses were negative. Reticulocytosis, spherocytes, increased osmotic fragility test, and persistent subclin‐ ical hemolysis indicated at transient aplastic crisis mimicking MDS-RAEB due to parvovirus infection in the setting of hereditary spherocytosis.

#### *2.1.2. Cytomegalovirus (CMV) infection*

• Differential diagnosis

50 Myelodysplastic Syndromes

myelodysplasia [17].

*2.1.1. Parvovirus infection*

infection.

• Conclusion and future recommendations

also give rise to dysmorphic findings in neutrophils.

closely mimick MDS will be delineated below.

**2.1. Viral and bacterial infections**

**2. Nonclonal disorders which present as dysplasia and cytopenia**

In a pilot and unpublished study that we carried on in our clinic, we compared the dysmorphic parameters in the neutrophils of patients with viral (n:6; infections: rubella, rubeola, viral eruption of unknown origin, Ebstain Barr virus infection), bacterial (n:7; infections: preseptal cellulitis, urinary infection, tonsillitis, maxillary sinusitis, lymphadenitis, otitis media) infections and those of healthy controls. We found that the neutrophil diameter of those with bacterial infections; the percentage of pseudo Pelger-Huet cells and irregular distribution of granules in both viral and bacterial infections; the percentage of chromatin clumping in viral diseases were higher than the control. These findings showed that nonspecific infections can

In another study, we reported that those with bacterial diseases additionally displayed comparable diameter, macropolycyte (neutrophils with diameter >14 μm) percentage, bizarre nucleus, irregular distribution of granules with those of pretreatment ITP who also displayed

Striking dyserytropoiesis was reported in tuberculosis [27]. Several viral infections which

In the literature, there are cases of parvovirus infection, with [19–21] or without [22] immu‐ nodeficiency or chronic hemolytic anemia which transiently or chronically mimicked MDS or

The reported case of Hasle et al. [19] was an 8-year-old, previously healthy boy who admitted to the hospital with severe anemia, moderate thrombocytopenia, and granulocytopenia and a 2 weeks' history of intermittent fever. Physical examination was normal except for pallor. Bone marrow was hypercellular with marked erythroblastopenia and maturation arrest of the erythropoietic cell line. No giant pronormoblast and hemophagocytosis was noted. Dysplasia in myeloid and megakaryocytic lineage was evident. He had increased immunoglobulin (Ig) M, low IgG, slightly decreased natural killer (NK) cells which reduced during follow-up;

The patient was assumed as MDS and was administered prednisolon, androgenic steroid, cyclophosphamid and cyclosporine, IgG infusion and frequent blood transfusions and developed hemochromatosis and hepatosplenomegaly. Thrombocytopenia deepened; hemoglobin transiently normalized. Parvovirus antibody studies revealed negative but when polymerase chain reaction technique became available, serum samples of the previous two years of the disease course and bone marrow smears were found positive for parvovirus

dyserythropoietic anemia [22]. Among them the two [19, 20] are of note.

impaired in vitro proliferation of blood mononuclear cells on stimulation.

Miyahara et al. [23] reported a 41-year-old, previously healthy man who developed severe thrombocytopenia with myelodysplastic changes of bone marrow and multiple autoimmune abnormalities, low CD4/CD8 ratio following CMV infection. The bone marrow aspiration was hypocellular with decreased megakaryocytes, atypical lymphocytes, and trilineage dysplasia. After a short-course prednisolone therapy, he improved.

It was suggested that direct CD34+ multipotent stem cells were infected with CMV giving rise to injury to the bone marrow cells. The inhibitory effect of cytokines [tumor necrosis factor alpha (TNF-α), interferon gamma (IF-)] produced by CMV-infected leukocytes and stromal cells on hematopoiesis and autoimmunity might have been responsible for myelodysplastic changes and thrombocytopenia.

#### *2.1.3. Human immunodeficiency virus (HIV) infection*

At the time of primary infection, transient pancytopenia, lymphocytosis, increased hemato‐ gones, increase in CD8+ lymphocytes, isolated autoimmune thrombocytopenia, anemia, reticulocytopenia, neutropenia, trilineage myelodysplasia both in the peripheral blood and bone marrow were reported. Megakaryocytes which were in normal or increased numbers showed apparent naked nuclei and were occasionally dysplastic [18]. Dysplastic findings were found increased and erythropoiesis became megaloblastic during antiretroviral therapy.

#### *2.1.4. Hepatitis C virus (HCV) infection*

HCV-infected patients frequently had varying degrees of bone marrow dysplasia and patients with pancytopenia were those who had the most frequent bone marrow abnormalities. In the cohort of HCV-infected patients, those with hematopoietic malignancy also existed [24]. However, bone marrow was a site where HCV replicated extrahepatically which contributed to the etiology of HCV-associated neutropenia and thrombocytopenia. Peripheral clearance or consumption of platelets might have increased in HCV infection also [75, 76] like in other abnormalities in infections [77].

#### *2.1.5. Virus infections in MDS*

It should not be forgotten that more than 50% of patients with myelodysplasia and chronic myeloproliferative diseases showed elevated antibody titers against viruses like EBV and HHV-6 [78].

#### **2.2. Parasitic infections**

#### *2.2.1. Visceral leishmaniasis*

Yaralı et al. [25] reported seven cases with leishmaniasis all of whom had pancytopenia, dysplasia in erythroid myeloid, and megakaryocytic lineages. The all qualitative and quanti‐ tative findings disappeared after 2 months' therapy.

The authors postulated that increased TNF-α which was shown to be associated with increased macrophages, increased oxidized pyrimidine nucleotides, decreased glutathione concentra‐ tion and presumably reduced clearance of free oxygen radicals might be responsible for myelodysplasia in visceral leishmaniasis, and other hematological findings.

Dhingra et al. [26] also reported 18 cases with leishmaniasis who had various combinations of cytopenia with increased bone marrow cellularity. Trilineage myelodysplasia (22%), bone marrow fibrosis (16.6%), hemophagocytosis (11.1%), and increased iron stores (33.3%) were evident.

It was thought that infected bone marrow stromal macrophages with leishmania, selectively enhanced myelopoiesis by granulocyte macrophage colony stimulating factor (GM-CSF) and TNF-α overproduction, giving rise to hypercellularity and trilineage myelodysplasia. In‐ creased iron stores were attributed to cytokine overproduction which also led to anemia.

#### *2.2.2. Others*

Secondary myelodysplasia due to plasmodium falciparum and *P. vivax* infection was also reported [27].

#### **2.3. Autoimmune disorders**

#### *2.3.1. Juvenile rheumatoid arthritis (JRA)*

Yetgin et al. [28] reported myelodysplasia in 17 patients with JRA, none of whom had received iron, corticosteroids, immunosuppressive drugs, or any transfusions, and none had acute infection or gross bleeding. Bone marrow of all cases revealed normal along with abnormal maturation at different levels, like left shift, along with trilineage dysplasia, the most promi‐ nent dysplasia being in myeloid lineage. Increased bone marrow cellularity, fatty changes, erythroid hypoplasia, myeloid and mild-moderate megakaryocytic hyperplasia were detected. They all had anemia, mostly being microcytic; the most had leukocytosis and thrombocytosis.

consumption of platelets might have increased in HCV infection also [75, 76] like in other

It should not be forgotten that more than 50% of patients with myelodysplasia and chronic myeloproliferative diseases showed elevated antibody titers against viruses like EBV and

Yaralı et al. [25] reported seven cases with leishmaniasis all of whom had pancytopenia, dysplasia in erythroid myeloid, and megakaryocytic lineages. The all qualitative and quanti‐

The authors postulated that increased TNF-α which was shown to be associated with increased macrophages, increased oxidized pyrimidine nucleotides, decreased glutathione concentra‐ tion and presumably reduced clearance of free oxygen radicals might be responsible for

Dhingra et al. [26] also reported 18 cases with leishmaniasis who had various combinations of cytopenia with increased bone marrow cellularity. Trilineage myelodysplasia (22%), bone marrow fibrosis (16.6%), hemophagocytosis (11.1%), and increased iron stores (33.3%) were

It was thought that infected bone marrow stromal macrophages with leishmania, selectively enhanced myelopoiesis by granulocyte macrophage colony stimulating factor (GM-CSF) and TNF-α overproduction, giving rise to hypercellularity and trilineage myelodysplasia. In‐ creased iron stores were attributed to cytokine overproduction which also led to anemia.

Secondary myelodysplasia due to plasmodium falciparum and *P. vivax* infection was also

Yetgin et al. [28] reported myelodysplasia in 17 patients with JRA, none of whom had received iron, corticosteroids, immunosuppressive drugs, or any transfusions, and none had acute infection or gross bleeding. Bone marrow of all cases revealed normal along with abnormal maturation at different levels, like left shift, along with trilineage dysplasia, the most promi‐ nent dysplasia being in myeloid lineage. Increased bone marrow cellularity, fatty changes,

myelodysplasia in visceral leishmaniasis, and other hematological findings.

abnormalities in infections [77].

*2.1.5. Virus infections in MDS*

**2.2. Parasitic infections**

*2.2.1. Visceral leishmaniasis*

tative findings disappeared after 2 months' therapy.

HHV-6 [78].

52 Myelodysplastic Syndromes

evident.

*2.2.2. Others*

reported [27].

**2.3. Autoimmune disorders**

*2.3.1. Juvenile rheumatoid arthritis (JRA)*

**Figure 1.** Dysmorphic hematological features of the peripheric blood smears of the patient ASİ with chronic ITP (a–g), patient MEY with JRA (h–n), patient TÇ with JRA (o–x). Courtesy of Turk J Med Sci [79]. **Neutrophils**: Macropolycytes (neutrophil > 15 μm) (b, c, h, i), hypersegmentation (c), cytoplasmic vacuoles (a), hypogranulation (d, o), cytoplasmic protrusions with or without granules (h, k), irregular distribution of granules (a, c, j), abnormal nuclei with nucleic protrusions (q, r), neutrophils with long chromatin between the nuclei (j, o), pseudo Pelger-Huet cells (o, p) **Lympho‐ cytes**: Cytoplasmic protrusions (e, n, u, v, w). Basophils: Centralization of granules (f), abnormal nuclei and hypogra‐ nulation (x). **Monocytes**: Abnormal nuclei (g, s, t), cytoplasmic vacuoles (g, s), cytoplasmic protrusion (g). **Platelets**: Big or giant platelets (l, m) **β-gal staining photographs of the patients**. Patient TÇ JRA (y), patient MBY with JRA (z), patient KÇ with SLE (aa), patient HA with acute ITP (ab), patient ASİ with chronic ITP (ac), control (ad) (×100).

The score of myelodysplastic peripheral blood findings but not those of bone marrow corre‐ lated significantly with CRP and ferritin.

It was postulated that abnormally regulated cytokines and other local intracellular messengers by cellular immune system lead to alterations of the microenvironment of bone marrow giving rise to the myelodysplastic features (**Figure 1**).

On the other hand, clinicians should keep cautious since rheumatoid arthritis and other rheumatoid disorders may present as a part of immune abnormalities in MDS also [72–74].

#### *2.3.2. Polyarteritis nodosa (PAN)*

Yetgin et al. [29] reported a child with hematopoietic dysplastic characteristics in an 11-yearold girl with PAN. While her blood smear revealed occasional trilineage dysplasia, her bone marrow displayed moderate cellularity, fatty changes, and trilineage dysplasia in addition to blast and blast-like mononuclear cells. She received therapy of methyl prednisolone and cyclophosphamide and all of the hematologic abnormalities were found to have resolved after 6 months. It was suggested that these dysplastic findings were associated with the primary inflammatory process and increased cytokines.

#### *2.3.3. Systemic lupus erythematosis (SLE)*

Voulgarelis et al. [30] reported bone marrow biopsy and aspiration findings of 40 SLE cases in comparison with 10 MDS-refractory anemia (RA) cases. The patients had mono-bi- or triline‐ age cytopenia. The bone marrows were hyponormocellular with increased erythroid and megakaryocytic lineages in the majority of cases. All patients had dyserythropoiesis and dysmegakaryopoiesis. Dysmyelopoiesis was less striking with a left shifting. While the rate of dyserythropoiesis and dysmegakaryopoiesis were similar in patients with SLE and MDS-RA (100% vs 100%), the features of bone marrow biopsy specimens differed in that normohypercellularity and abnormal localization of immature progenitors (ALIP) aggregates were less but bone marrow necrosis was higher in SLE. Dilated sinuses (20%) were seen in SLE while no dilated sinus was noted in MDS-RA (0%). Increased reticulin, striking stromal edema, lack of inflammatory vascular damage and lack of microvascular obstruction by thrombus plugs, aggregates of T and B lymphocytes with polyclonal immunoglobulin expression were other striking features of SLE [30]. Specific lupus erythematosus (LE) cells (neutrophils containing a round, amorphous mass of purple, degraded nuclear material) were reported to be rarely seen when the bone marrow aspirate is anticoagulated and spreading of films were delayed [27]. These findings showed that bone marrow was a main target in SLE.

In SLE, it was shown that bone marrow fibroblasts could not produce enough hematopoietic growth factors and stromal cells of SLE patients failed to support allogenic progenitor cell growth in culture leading to defective hematopoietic microenvironment and altered cytokine expression. Additionally, autoreactive lymphocytes in the bone marrow of SLE patients might have directly caused immune destruction of both stromal cells and hematopoietic cells and indirectly affected them via releasing pro-inflammatory cytokines like TNF-α [35]. Secondary dysplastic changes in autoimmune diseases, in particular SLE, were demonstrated to closely mimick those in HIV [27] (**Figure 1**).


\*Complete thrombocyte response in 50% (at 3 months) and 33% (at 12–54 months) of patients with short thrombocyte lifespan (<3.5 days), no transfusion requirement, but sustained neutropenia [91, 92].

**Table 5.** Characteristics of chronic ITP and MDS with unilineage cytopenia as thrombocytopenia (RCC subgroup of childhood MDS and RCUD, RCMD in adulthood MDS).

#### *2.3.4. Immune thrombocytopenic purpura*

It was postulated that abnormally regulated cytokines and other local intracellular messengers by cellular immune system lead to alterations of the microenvironment of bone marrow giving

On the other hand, clinicians should keep cautious since rheumatoid arthritis and other rheumatoid disorders may present as a part of immune abnormalities in MDS also [72–74].

Yetgin et al. [29] reported a child with hematopoietic dysplastic characteristics in an 11-yearold girl with PAN. While her blood smear revealed occasional trilineage dysplasia, her bone marrow displayed moderate cellularity, fatty changes, and trilineage dysplasia in addition to blast and blast-like mononuclear cells. She received therapy of methyl prednisolone and cyclophosphamide and all of the hematologic abnormalities were found to have resolved after 6 months. It was suggested that these dysplastic findings were associated with the primary

Voulgarelis et al. [30] reported bone marrow biopsy and aspiration findings of 40 SLE cases in comparison with 10 MDS-refractory anemia (RA) cases. The patients had mono-bi- or triline‐ age cytopenia. The bone marrows were hyponormocellular with increased erythroid and megakaryocytic lineages in the majority of cases. All patients had dyserythropoiesis and dysmegakaryopoiesis. Dysmyelopoiesis was less striking with a left shifting. While the rate of dyserythropoiesis and dysmegakaryopoiesis were similar in patients with SLE and MDS-RA (100% vs 100%), the features of bone marrow biopsy specimens differed in that normohypercellularity and abnormal localization of immature progenitors (ALIP) aggregates were less but bone marrow necrosis was higher in SLE. Dilated sinuses (20%) were seen in SLE while no dilated sinus was noted in MDS-RA (0%). Increased reticulin, striking stromal edema, lack of inflammatory vascular damage and lack of microvascular obstruction by thrombus plugs, aggregates of T and B lymphocytes with polyclonal immunoglobulin expression were other striking features of SLE [30]. Specific lupus erythematosus (LE) cells (neutrophils containing a round, amorphous mass of purple, degraded nuclear material) were reported to be rarely seen when the bone marrow aspirate is anticoagulated and spreading of films were delayed

In SLE, it was shown that bone marrow fibroblasts could not produce enough hematopoietic growth factors and stromal cells of SLE patients failed to support allogenic progenitor cell growth in culture leading to defective hematopoietic microenvironment and altered cytokine expression. Additionally, autoreactive lymphocytes in the bone marrow of SLE patients might have directly caused immune destruction of both stromal cells and hematopoietic cells and indirectly affected them via releasing pro-inflammatory cytokines like TNF-α [35]. Secondary dysplastic changes in autoimmune diseases, in particular SLE, were demonstrated to closely

[27]. These findings showed that bone marrow was a main target in SLE.

rise to the myelodysplastic features (**Figure 1**).

inflammatory process and increased cytokines.

*2.3.3. Systemic lupus erythematosis (SLE)*

mimick those in HIV [27] (**Figure 1**).

*2.3.2. Polyarteritis nodosa (PAN)*

54 Myelodysplastic Syndromes

In a previous study, we established neutrophil and eosinophil dysmorphism and increased macropolycytes in patients with acute and chronic ITP before treatment, in comparison with normal children. Several dysplastic features increased at the end of mega dose steroid (methyl prednisolone 30 mg/kg/day × 3 days followed by 20 mg/kg/day for the consecutive 4 days) therapy but decreased within 1–4 weeks after therapy was stopped. Hyperdiploidy in neutrophils which developed during steroid therapy normalized 7 days after therapy was stopped [17]. Dysplastic features were noted in other cell lines too [79].

These findings suggested that not only an intrinsic megakaryocyte proliferative defect giving rise to deficient platelet production were present in refractory chronic ITP patients but a defect before or at the level of colony forming unit-granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM) also. The antiplatelet antibodies and the increased cytokines in ITP [80] might have been effective at this level (**Figure 1**).

#### *2.3.4.1. Differentiation between MDS (refractory thrombocytopenia) and chronic ITP*

Myelodysplastic syndrome with isolated thrombocytopenia (RCC subgroup of childhood MDS and RCUD, RCMD, MDS-U subgroups of adulthood MDS) can be masqueraded as refractory chronic ITP and the accurate diagnosis may be challenging due to close similarities between the two entities like decreased life span, decreased production rate in thrombocytes, increased thrombocyte destruction, presence of dysplastic findings in myeloid and megakar‐ yocytic cell lines including micromegakaryocytes, naked megakaryocyte nuclei and megakar‐ yocyte emperipolesis; and additionally megakaryocyte apoptosis, platelet microparticles and good response to splenectomy in several patients [17, 31, 81–93] (**Table 5**).

**Figure 2.** Myelodysplastic findings in a patient with MDS (refractory thrombocytopenia). Courtesy of Ped Hemat On‐ col [31]. **Erythroid serie**: Interchromatin bridge between erythroblasts (a), spherocytes (b). **Neutrophilic serie**: Bizarre nucleus, nuclei with striking chromatin clumping, abnormal projections, cytoplasm with irregular distribution of gran‐ ules and vacuoles (c), nucleocytoplasmic asyncrony (c, d). **Eosinophilic serie** with micronuclei, cytoplasmic vacuola‐ tion, cytoplasm with both eosinophilic and basophilic granules (e). **Monocytic serie** with cytoplasmic vacuoles (f). **Blast-like cells** (g). **Histiocytic serie**: Sea-blue histiocyte (h). **Mitotic cells**: Mitosis in an unknown cell (i). **Apoptotic cells** with condensed and fragmented nuclei and condensed cytoplasm (j).

Thrombocyte microparticles per thrombocyte were reported more than normal in both disorders [90] and the both can benefit from splenectomy although with different success rates [91–93]. Two of these characteristics can be used to differentiate between the two entities. The first is that, while megakaryocyte apoptosis in ITP starts at stage 3 (mature) megakaryocyte level [82] (or apoptosis does not take place in megakaryocytes of ITP patients [85], megakar‐ yocyte apoptosis in MDS is detected in micromegakaryocyte level [31, 83]. The second is that while apoptosis takes place in myeloid, lymphoid, and monocytic cell lines in MDS [86, 87], no increase in apoptosis (in granulocytes and lymphocytes) on the contrary resistance to apoptosis (in lymphocytes) [88, 89] were reported in ITP (**Table 5**). We followed a patient with RCC that mimicked therapy resistant chronic ITP, who developed intracranial hemorrhage twice but underwent a successful bone marrow transplantation [31] (**Figures 2**–**4**).

*2.3.4.1. Differentiation between MDS (refractory thrombocytopenia) and chronic ITP*

56 Myelodysplastic Syndromes

good response to splenectomy in several patients [17, 31, 81–93] (**Table 5**).

Myelodysplastic syndrome with isolated thrombocytopenia (RCC subgroup of childhood MDS and RCUD, RCMD, MDS-U subgroups of adulthood MDS) can be masqueraded as refractory chronic ITP and the accurate diagnosis may be challenging due to close similarities between the two entities like decreased life span, decreased production rate in thrombocytes, increased thrombocyte destruction, presence of dysplastic findings in myeloid and megakar‐ yocytic cell lines including micromegakaryocytes, naked megakaryocyte nuclei and megakar‐ yocyte emperipolesis; and additionally megakaryocyte apoptosis, platelet microparticles and

**Figure 2.** Myelodysplastic findings in a patient with MDS (refractory thrombocytopenia). Courtesy of Ped Hemat On‐ col [31]. **Erythroid serie**: Interchromatin bridge between erythroblasts (a), spherocytes (b). **Neutrophilic serie**: Bizarre nucleus, nuclei with striking chromatin clumping, abnormal projections, cytoplasm with irregular distribution of gran‐ ules and vacuoles (c), nucleocytoplasmic asyncrony (c, d). **Eosinophilic serie** with micronuclei, cytoplasmic vacuola‐ tion, cytoplasm with both eosinophilic and basophilic granules (e). **Monocytic serie** with cytoplasmic vacuoles (f). **Blast-like cells** (g). **Histiocytic serie**: Sea-blue histiocyte (h). **Mitotic cells**: Mitosis in an unknown cell (i). **Apoptotic**

Thrombocyte microparticles per thrombocyte were reported more than normal in both disorders [90] and the both can benefit from splenectomy although with different success rates [91–93]. Two of these characteristics can be used to differentiate between the two entities. The first is that, while megakaryocyte apoptosis in ITP starts at stage 3 (mature) megakaryocyte

**cells** with condensed and fragmented nuclei and condensed cytoplasm (j).

**Figure 3.** Dysmorphism in megakaryocytic serie, in a patient with MDS (refractory thrombocytopenia). Courtesy of Ped Hemat Oncol [31]. **Evaluation by light microscopy**: Mononuclear megakaryocytes (a, b), megakaryocyte with ring-shaped nucleus (b), megakaryocytes with nuclei that are being extruded out of the cell (i–k, o), the cytoplasm which is lobulated (i), basophilic and condensed (i, k), naked megakaryocyte nuclei (f–h) with abnormal nuclear shape (g–i), macroplatelets (c–e, m), dysmorphic platelets (e, l, m) are seen (×100). **Evaluation by transmission electron mi‐ croscopy**: Stage I megakaryocyte with large, oval, and intended nucleus and a cytoplasm containing abundant ribo‐ somes and granules. Demarcation system of membranes and granules are abundant in cytoplasm, all of which indicates nucleocytoplasmic asyncrony. The granules were identified as azurophilic granules (single arrow) and un‐ identifiable, large, oval, and electron lucent, abnormal granules (double arrow) (×10,000) (p) and lots of free ribosomes, azurophilic (single arrow) and abnormal unidentifiable, large, oval, electron lucent granules (double arrow) with de‐ marcation membranes in the cytoplasm of the same cell (×27,800) (q). A megakaryocyte that shows abundant demarca‐ tion membranes, ribosomes, and granules. The granules are heterogenous as to both size and electron density. A phagosome (emperipolesis) is also seen (×12,930) (r). A stage I megakaryocyte with double nucleoli and abundant de‐ marcation membranes, abundant azurophilic (single arrow) and unidentifiable, large, oval, electron lucent abnormal granules (double arrow) (s). Apoptotic stage I megakaryocyte that shows a condensed nuclear fragment and con‐ densed cytoplasm, but mitochondria, mitochondrial crystae, and demarcation membranes are still intact (×16,700) (t).

**Figure 4.** Fas, annexin and bcl-2 values in lymphocytes (R1), monocytes (R2), granulocytes (R3) from the bone marrow of the child with MDS (refractory thrombocytopenia), established by flow cytometry (Coulter Elite). Courtesy of Ped Hemat Oncol [31].

#### *2.3.5. Autoimmune neutropenia*

Although chronic idiopathic and autoimmune neutropenia are considered as benign disorders, it was reported that the bone marrow of these patients displayed dyserythropoiesis by 55% and they transformed to clonal hematological diseases including NK expansion, hairy cell leukemia, myelomonocytic leukemia, and MDS (RCUD, RCMD) within 30 months, with a rate up to 6.5%. Therefore, these patients should be closely followed up [32].

#### **2.4. Hemophagocytic lymphohistiosis (HLH, hemophagocytic syndrome)**

All patients whom we followed in our clinic due to primary or secondary HLH had myelo‐ dysplastic features in addition to cytopenia involving at least two cell lines. The common findings in the bone marrow were erythroid hyperplasia, chromatin bridges between erythroid precursors, multiple nuclei in erythroblasts, mild megaloblastic changes, vacuoles in erythro‐ blasts, myeloid precursors and monocytes, sometimes in thrombocytes; hypogranulation in neutrophil myelocytes, micromyelocytes, irregular distribution of cytoplasmic granules, large and sometimes hybrid granules in eosinophils; anisocytosis in thrombocytes including giant thrombocytes, naked megakaryocyte cytoplasm, megakaryocyte emperipolesis, oligomono‐ nuclear megakaryocytes. These changes were probably due to the cytokine storm that played the major role in the pathogenesis of HLH (**Figure 5**).

**Figure 4.** Fas, annexin and bcl-2 values in lymphocytes (R1), monocytes (R2), granulocytes (R3) from the bone marrow of the child with MDS (refractory thrombocytopenia), established by flow cytometry (Coulter Elite). Courtesy of Ped

Although chronic idiopathic and autoimmune neutropenia are considered as benign disorders, it was reported that the bone marrow of these patients displayed dyserythropoiesis by 55% and they transformed to clonal hematological diseases including NK expansion, hairy cell leukemia, myelomonocytic leukemia, and MDS (RCUD, RCMD) within 30 months, with a rate

All patients whom we followed in our clinic due to primary or secondary HLH had myelo‐ dysplastic features in addition to cytopenia involving at least two cell lines. The common

up to 6.5%. Therefore, these patients should be closely followed up [32].

**2.4. Hemophagocytic lymphohistiosis (HLH, hemophagocytic syndrome)**

Hemat Oncol [31].

58 Myelodysplastic Syndromes

*2.3.5. Autoimmune neutropenia*

**Figure 5.** Myelodysplastic bone marrow findings of two patients with secondary (a, b) and primary (c–o) hemophago‐ cytic histiocytosis (HLH) (personal archives). Megakaryocytes with mononuclear nuclei that have irregular edges and condensed cytoplasm are seen just after (a) and during (b) the process of extruding the nuclei out of the cell, develop‐ ing naked megakaryocyte cytoplasm and nucleus (a). Bilobed erythroid precursor (c), eosinophil myelocytes with cyto‐ plasmic vacuolation and rare basophilic granules, cytoplasmic protrusion (d), a naked nuclei of an unknown cell (e), cells or formations with heterogenous morphology consisting of numerous vacuoles with various sizes (f, g, i), indis‐ tinguishable from abnormal trombocytes and detached cytoplasm of hemophagocyting histiocyte (h), hypogranulated and vacuolated band (h), ringed nuclei in eosinophilic myelocytes (j, k), internuclear chromatin bridge (slightly dim) (l), cytoplasmic protrusions in basophilic erythroblasts (m), bilobed normoblast (n), cytoplasmic vacuolation in a proer‐ ythroblast (o) (vitamin B12, folic acid, Cu, Zn levels were normal).

#### **2.5. Nutritional deficiencies**

#### *2.5.1. Malnutrition*

Mono-, bi-, pancytopenia, and hematopoietic dysplasia were reported in patients with malnutrition, who were generally deficient in iron, vitamin B12, folate, and trace elements also. Bone marrow cellularity was reduced with normoblastic dyserythropoiesis. Giant metamye‐ locytes, vacuolation in erythroid and granulocytic precursors, abnormal sideroblasts including ring sideroblasts [27], hemophagocytosis, necrotic cells, and rarely dysmegakaryocytosis were reported.

**Figure 6.** Myelodysplastic bone marrow findings of a child with malnutrition and gelatinous transformation (personal archives). Basophilic erythroblasts with cytoplasmic protrusions (a) and vacuoles (b), agranular neutrophil (c, i) with numerous cytoplasmic vacuoles and hypersegmented nucleus (c), hypoagranular neutrophils with hypolobulated nu‐ clei with abnormal protrusions, mild chromatin clumping (d, h), eosinophil with abnormal nucleus, irregular distribu‐ tion of granules (g), eosinophilic band with few basophilic granules distributed irregularly (j), vacolated monocytes (e, j) and histiocytes (f), degenerating histiocytes (j), hypha and amorphous material representing the gelatinous material (i, k).

In anorexia nervosa, severe diseases with cachexia and starvation, acantocytosis in the peripheral blood, hypocellularity with/without "gelatinous transformation" were noted. In gelatinous transformation, the fat cells in the bone marrow are lost, and hematopoietic cells are replaced by extracellular matrix material composed of acid mucopolysaccharide rich in hyaluronic acid is detected [18, 33]. All the reported cases had anemia, leukopenia ± throm‐ bocytopenia [33, 34], rarely only anemia [35] (**Figure 6**).

Trilineage dysplasia in peripheric blood of iron deficient patients were reported, being higher than the control group. In addition, microspherocytes which were observed in 20% of irondeficient patients were not noted in the control group [36]. Leukopenia and thrombocytopenia may accompanied these changes as iron deficiency deepened, making the differential diag‐ nosis between iron deficiency anemia and MDS more difficult. Alcantara et al. [94] showed that iron supported 11 genes in phorbol myristate acetate-induced HL-60 cell lines which were involved in critical cell decision points to pursue a differentiation or cell death pathway.

Additionally, increased erythropoietin in iron deficiency anemia might also have activated hematopoietic lineages. Presence of microspherocytes was attributed to a putative involve‐ ment of Rac1 and Rac2 GTPase, also known as Ras-related C3 botulinum toxin substrates 1 and 2 GTPase deficiency which reportedly altered actin assembly in red cells in mice. Further factors like cellular metabolic enzyme changes, cell growth and differentiation, and gene expression regulation might have been involved in the pathogenesis [36].

On the other hand, leukemia cases who were under chemotherapy and had myelodysplastic features were shown to have hypochromic macrocytes and increased serum iron and ferritin levels and increased soluble transferrin receptor implying at a disturbance in utilizing iron (functional iron deficiency) [49].

#### *2.5.2. Megaloblastic anemia*

See Section 4.2.

**2.5. Nutritional deficiencies**

Mono-, bi-, pancytopenia, and hematopoietic dysplasia were reported in patients with malnutrition, who were generally deficient in iron, vitamin B12, folate, and trace elements also. Bone marrow cellularity was reduced with normoblastic dyserythropoiesis. Giant metamye‐ locytes, vacuolation in erythroid and granulocytic precursors, abnormal sideroblasts including ring sideroblasts [27], hemophagocytosis, necrotic cells, and rarely dysmegakaryocytosis were

**Figure 6.** Myelodysplastic bone marrow findings of a child with malnutrition and gelatinous transformation (personal archives). Basophilic erythroblasts with cytoplasmic protrusions (a) and vacuoles (b), agranular neutrophil (c, i) with numerous cytoplasmic vacuoles and hypersegmented nucleus (c), hypoagranular neutrophils with hypolobulated nu‐ clei with abnormal protrusions, mild chromatin clumping (d, h), eosinophil with abnormal nucleus, irregular distribu‐ tion of granules (g), eosinophilic band with few basophilic granules distributed irregularly (j), vacolated monocytes (e, j) and histiocytes (f), degenerating histiocytes (j), hypha and amorphous material representing the gelatinous material

*2.5.1. Malnutrition*

60 Myelodysplastic Syndromes

reported.

(i, k).

#### *2.5.3. Copper deficiency*

Copper is a cofactor of a number of enzymes (cuproenzymes) including those important for hematologic system like hephaestin, seruloplasmin (ferroxidases) and others like cytochrome *c* oxidase, superoxide dismutase 1, extracellular superoxide dismutase, and zyklopen, a new member of the vertebrate multicopper ferroxidase family

Pregnant and lactating women, premature infants, those with malabsorption, inflammatory bowel diseases, celiac disease, long lasting diarrhea, short bowel syndrome, those under total parenteral nutrition, or nutrition through jejunal tube, those who underwent gastric resection, bariatric surgery; Wilsons' disease patients who consumed copper-depleting drugs, conditions with excess zinc are under risk of copper deficiency [37–41].

The most important problem in copper deficiency lays in its diagnosis. It may be misdiagnosed as MDS or underdiagnosed. The time which lapses until appropriate diagnosis is made was reported as approximately 1 year. Early diagnosis is important since therapy after neurological symptoms have developed is difficult [40].

On the other hand, it is of note that 11 out of 32 MDS patients were found to have copper deficiency [95].

The most striking hematologic findings in copper deficiency are mono-, bi-, and pancytopenia [39–41]. Anemia which is the most common hematologic finding (97.5%) in copper deficiency is generally normochromic or macrocytic but rarely microcytic, being dependent on the severity of the deficiency. When the activity of copper-dependent enzymes decrease, iron absorption is expected to be impaired, iron transport across intestinal cells be decreased, conversion of ferrous iron to the ferric form which is necessary for transport of iron by transferrin be impaired, conversion of ferric iron to ferrous iron which is necessary for incorporation of iron into the protoporphyrin molecule during hemoglobin synthesis be inadequate. The latter defect gives rise to both formation of ring sideroblasts and possibly erythrocyte membrane defect due to low levels of antioxidant zinc/copper dismutase activity necessary to convert superoxide-free radicals to hydrogen peroxide [37, 39]. However, the mechanism of anemia is not fully understood [37].


**Table 6.** Bone marrow findings in copper deficiency in comparison with MDS.

Leukopenia in copper deficiency is together with neutropenia which was reported to be the most frequent and earliest manifestation of copper deficiency [41]. The neutrophils in pe‐ ripheric blood smear are dysplastic. Impaired and delayed maturation, differentiation and regeneration of hematopoietic precursor cells, increased destruction of myeloid precursors in the bone marrow, defective neutrophil egress from the bone marrow, shortened life-span of neutrophils, and presence of antibodies to neutrophils are the possible etiologic factors for neutropenia [39, 41].

The bone marrow is generally hypercellular [40] with increased myeloid and/or erythroid precursors mimicking myeloid and erythroid arrest, vacuolization in erythroid and myeloid precursors, increased iron stores, prominent ring sideroblasts, plasma cells in which hemosi‐ derin is incorporated, increased hematogones [37] with [37, 38], without [39] myelodysplasia. In MDS, generally erythroid hyperplasia [15] is encountered. Myeloid arrest or left shift in granulopoiesis is seen generally in RAEB subgroup of MDS [96]. Characteristics of MDS and copper deficiency are summarized in **Table 6**.

#### *2.5.4. Vitamin D deficiency*

reported as approximately 1 year. Early diagnosis is important since therapy after neurological

On the other hand, it is of note that 11 out of 32 MDS patients were found to have copper

The most striking hematologic findings in copper deficiency are mono-, bi-, and pancytopenia [39–41]. Anemia which is the most common hematologic finding (97.5%) in copper deficiency is generally normochromic or macrocytic but rarely microcytic, being dependent on the severity of the deficiency. When the activity of copper-dependent enzymes decrease, iron absorption is expected to be impaired, iron transport across intestinal cells be decreased, conversion of ferrous iron to the ferric form which is necessary for transport of iron by transferrin be impaired, conversion of ferric iron to ferrous iron which is necessary for incorporation of iron into the protoporphyrin molecule during hemoglobin synthesis be inadequate. The latter defect gives rise to both formation of ring sideroblasts and possibly erythrocyte membrane defect due to low levels of antioxidant zinc/copper dismutase activity necessary to convert superoxide-free radicals to hydrogen peroxide [37, 39]. However, the

**Copper deficiency MDS**


5, 14, 39]

[96]





symptoms have developed is difficult [40].

mechanism of anemia is not fully understood [37].

Vacuolization -In erythroid + myeloid lineages [39]

Other -Left shift in myelopoiesis



Increased hematogones -Present [37] -Absent [39]

blast stage [37]

**Table 6.** Bone marrow findings in copper deficiency in comparison with MDS.



Ring sideroblast -Present [37–39] -Present only in RARS subgroup [2, 5]

Leukopenia in copper deficiency is together with neutropenia which was reported to be the most frequent and earliest manifestation of copper deficiency [41]. The neutrophils in pe‐ ripheric blood smear are dysplastic. Impaired and delayed maturation, differentiation and regeneration of hematopoietic precursor cells, increased destruction of myeloid precursors in the bone marrow, defective neutrophil egress from the bone marrow, shortened life-span of

deficiency [95].

62 Myelodysplastic Syndromes

Dysmegakaryopoiesis (nuclear lobulation and abnormal sizes)

Dysplasia -No [39]

Vitamin D has both proliferating and differentiating effect on hematopoiesis [42]. The bone marrow taken from infants with vitamin D deficiency rickets and anemia showed early signs of myelofibrosis with increase of reticulin which was reversed by vitamin D treatment [97]. Anemia, thrombocytopenia, hepatosplenomegaly, hypocellularity and increased osteoblast count in bone marrow, and hematopoietic precursors in spleen aspirates were striking. Hypochromia, macrocytosis, tear drop cells, young myeloid elements along with nucleated red blood cells were evident [98]. Dysdifferentiation due to its deficiency might have been aggravated by coexistent malnutrition in many of several patients.

#### *2.5.5. Hypervitaminosis A*

An infant with hypervitaminosis A reportedly had eversible severe anemia, thrombocytope‐ nia, and dyserythropoiesis. It was shown that in overdoses, vitamin A strongly inhibited the proliferation of multipotent hematopoietic cell line and bone marrow mesenchymal stem cells, through upregulating p21Cip1 and p27Kip1, cyclin-dependent kinase inhibitors [43].

#### **2.6. Severe congenital neutropenia (SCN)**

We, previously detected hematopoietic dysmorphism in congenital neutropenia, their nonneutropenic parents and one sibling, irrespective to the neutropenia mutation that the patients had [45, 99]. All the tested patients were negative for molecular genetics of MDS and were normal in conventional cytogenetics. Apoptosis of lymphocytes, granulocytes [45, 99], and monocytes [45], of both patients and parents and rapid cell senescence (RCS) in leukocytes of a few patients and their mothers were established [45, 99]. A substantial portion of cases had clinical or laboratory evidence of hemorrhagic diathesis and low NK and CD4+ cells.

These findings showed that pluripotent stem cells were involved in SCN irrespective to the genetic defect and non-neutropenic family members were also affected ([45, 99], study in submission) and congenital neutropenia and MDS shared the same death types and involved pluripotent stem cells.On the other hand, it should not be forgotten that MDS can present as isolated neutropenia (RCC, as refractory neutropenia). In our clinic, we followed a 4-year-old girl who was admitted to our hospital for chronic neutropenia, but the genetic evaluation revealed trisomy 8 and a complex karyotype; while the molecular genetic studies for congenital neutropenia (*HAX1, ELANE, and G6PC3*) were negative (unpublished data).

**Figure 7.** Myelodysplastic bone marrow findings of a 5-year-old patient with ALL who was on chemotherapy and had coexistent autoimmune hemolytic anemia [100] (personal archives). A Gaucher-like histiocyte that is hemophagocytos‐ ing a cell (a), internuclear chromatin bridge between two erythroblasts (b) (arrow), multinucleated erythroblasts with various sizes (c, e, g, h), striking megaloblastic changes (d), basophilic stippling (b, e–h).

#### **2.7. Inherited conditions**

revealed trisomy 8 and a complex karyotype; while the molecular genetic studies for congenital

**Figure 7.** Myelodysplastic bone marrow findings of a 5-year-old patient with ALL who was on chemotherapy and had coexistent autoimmune hemolytic anemia [100] (personal archives). A Gaucher-like histiocyte that is hemophagocytos‐ ing a cell (a), internuclear chromatin bridge between two erythroblasts (b) (arrow), multinucleated erythroblasts with

various sizes (c, e, g, h), striking megaloblastic changes (d), basophilic stippling (b, e–h).

neutropenia (*HAX1, ELANE, and G6PC3*) were negative (unpublished data).

64 Myelodysplastic Syndromes

Özbek et al. [46] reported myelodysplastic features in myeloid and erythroid cell lines, in 20 patients with microdeletion 22q11.2 (del22q11.2) with slight cytopenia. Their smears showed dysmorphism in erythroid and myeloid cell lineages, in addition to a few vacuolated plasma‐ toid lymphocytes; monocytic cells mimicking hypogranular myelocytes with cytoplasmic vacuoles and protrusions and blast-like cells.

Myelodysplasia scores in the myeloid cells and eosinophils and macropolycyte percentages were higher than those with conotruncal heart defects, viral and bacterial infections, and healthy children. Genes in the deleted region, like human cell division cycle-related (hCDCrel) gene was proposed to be responsible for these changes.

Other inherited conditions which present as dyserythropiesis and anemia are congenital dyserythropoietic anemia, thalassemia, congenital dyserythropoietic porphyria, mitochondri‐ al myopathies, hereditary sideroblastic anemia, homozygous hemoglobin C, heterozygous unstable hemoglobins, some cases with thiamine-responsive anemia with diabetes and deafness, homozygote pyruvate kinase deficiency, stress erythropoiesis like severe hemolytic anemia [100] (**Figure 7**).

Those who present as dysgranulopoiesis with/without neutropenia are mitochondrial cytopaties, myelokathexis, and congenital neutropenia [45]. Those with dysmegakaryopoiesis and thrombocytopenia are inherited thrombocytopenias. Patients with GATA1 mutations have anemia and neutropenia together with trilineage dysplasia [27]. Patients with mevalonc aciduria due to mevalonate kinase deficiency have anemia, thrombocytopenia with/without fluctuation, dysplasia in erythroid and myeloid lineages [47].

#### **2.8. Malignant lymphoma**

In non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) patients, myelodysplasia in granulocytic and erythroid lineages were noted without any marked myelodysplasia in megakaryocytic lineage. The myelodyspastic features were found comparable in patients with and without bone marrow infiltration of lymphoma cells.

The bone marrow was normal or hypercellular, with normal, reduced number of erythroid and increased number of myeloid cells, normal, or increased megakaryocytes; ALIP was not encountered. Reticulin fibrosis was rare (6.1%).

The myelodysplasia in lymphoma was thought to be a reaction to the lymphoma or to result from an impaired bone marrow stem cell [48].

#### **2.9. Effect of drugs and toxins**

#### *2.9.1. Chemotherapy*

Most of chemotherapeutic and immunosuppressive agents give damage to the bone marrow, inducing megaloblastic dyserythropoiesis in low doses, and hypoplasia in high doses. Drugs that cause megaloblastosis are methotrexate, cyclophosphamide, daunorubicin, doxorubicin, cytarabine, hydroxyurea, azathioprine, and zidovudine [27]. Mycophenolate mofetil is known to cause Pelger-Huet anomaly, abnormal chromatin clumping, detached nuclear fragments in granulocyte lineage. Alemtuzumab was also reported to be associated with increased dys‐ plastic features and virus-related hemophagocytic syndrome [27]. In our experience, the most consistent finding of dysplasia in patients who receive chemotherapy was hypoagranulation of myeloid cells (**Figures 7** and **8**).

We previously showed that leukemia patients displayed hypochromic macrocytes in their peripheric blood due to failure to utilize iron ([49], study in submission). Additionally, serum reticulocyte counts in the beginning of chemotherapy blocks declined significantly in the end of the blocks when erythropoiesis was markedly depressed and were found to have increased significantly at the beginning of the next chemotherapy block when the bone marrow regen‐ erated and erythropoiesis increased [49].

Increased apoptosis, increased hemophagocyting macrophages, erythroid and megakaryo‐ cytic regeneration generally preceding granulocytic regeneration, megakaryocytic clustering and ALIP together with myelodysplasia were described after intensive chemotherapy and persisted for months [27]. The infections that the patients could have developed, possibly aggravated the myelodysplasia.

**Figure 8.** Neutrophils of a pediatric ALL patient while he was on maintenance chemotherapy (a, b), and 6 months after cessation of therapy (c, d) (personal archives). Cytoplasmic agranulation (a–d), large size (a–d), chromatin clumping (b, c), long chromatin string between the nuclear lobes (d) are striking.

On the other hand, these findings closely overlap with those in therapy-related MDS (t-MDS) or therapy-related myeloid neoplasms (t-MN) in the new nomenclature. It was reported that t-MN followed treatment of lymphomas and solid tumors but more rarely leukemias [101]. Appearance of new dysplastic changes after complete remission of leukemia [102] or solid tumors should alert the physician for development of t-MN.

#### *2.9.2. Steroids*

that cause megaloblastosis are methotrexate, cyclophosphamide, daunorubicin, doxorubicin, cytarabine, hydroxyurea, azathioprine, and zidovudine [27]. Mycophenolate mofetil is known to cause Pelger-Huet anomaly, abnormal chromatin clumping, detached nuclear fragments in granulocyte lineage. Alemtuzumab was also reported to be associated with increased dys‐ plastic features and virus-related hemophagocytic syndrome [27]. In our experience, the most consistent finding of dysplasia in patients who receive chemotherapy was hypoagranulation

We previously showed that leukemia patients displayed hypochromic macrocytes in their peripheric blood due to failure to utilize iron ([49], study in submission). Additionally, serum reticulocyte counts in the beginning of chemotherapy blocks declined significantly in the end of the blocks when erythropoiesis was markedly depressed and were found to have increased significantly at the beginning of the next chemotherapy block when the bone marrow regen‐

Increased apoptosis, increased hemophagocyting macrophages, erythroid and megakaryo‐ cytic regeneration generally preceding granulocytic regeneration, megakaryocytic clustering and ALIP together with myelodysplasia were described after intensive chemotherapy and persisted for months [27]. The infections that the patients could have developed, possibly

**Figure 8.** Neutrophils of a pediatric ALL patient while he was on maintenance chemotherapy (a, b), and 6 months after cessation of therapy (c, d) (personal archives). Cytoplasmic agranulation (a–d), large size (a–d), chromatin clumping (b,

On the other hand, these findings closely overlap with those in therapy-related MDS (t-MDS) or therapy-related myeloid neoplasms (t-MN) in the new nomenclature. It was reported that t-MN followed treatment of lymphomas and solid tumors but more rarely leukemias [101]. Appearance of new dysplastic changes after complete remission of leukemia [102] or solid

of myeloid cells (**Figures 7** and **8**).

66 Myelodysplastic Syndromes

erated and erythropoiesis increased [49].

c), long chromatin string between the nuclear lobes (d) are striking.

tumors should alert the physician for development of t-MN.

aggravated the myelodysplasia.

Steroids give rise to hyperdiploidy, and therefore macropolycytes in neutrophils [17] in addition to abnormal nuclear lobulation (**Figures 9** and **10**).

**Figure 9.** Peripheric blood neutrophils of children who were on high dose or long-term steroid therapy for acute and chronic ITP (personal archives). On the seventh (last) day of mega-dose methyl prednisolone therapy [116] of patient IY (a–g, i, j, l, o) and OI (k); during the phase of tapering down long-term prednisolone therapy of patient YP (n, h, p); 6 months after the last steroid therapy in YP (m). Macropolycytes (neutrophils with >14 μm diameter) in 15–20 μm diameter (a, c, h, m, n, p), pseudo-Pelger-Huet/like cells (a, c, j), chromatin clumping (c, f, k, n, p), bizarre nucleus (b, c, d, e, g, j, o), vacuolated eosinophil with both basophilic and eosinophilic granules (l).

#### *2.9.3. Alcohol*

Anemia with/without other cytopenias or pancytopenia were reported in alcohol dependent patients. Anemia was normochromic or macrocytic with round macrocytes (unlike oval macrocytes of megaloblastic anemia) [27, 50]. Vacuolated neutrophils, stomatocytes, sometimes target cells were evident. In hemolytic anemia and hyperlipidemia due to alcoholic liver disease, spherocytes, irregularly contracted cells (Zieve's syndrome) were demonstrated [27].

**Figure 10.** Various cell cycle configurations in patients with myelodysplasia (personal archives). Cell cycle analysis of granulocytic and mononuclear cells of children with acute or chronic immune thrombocytopenic purpura (ITP) before, during and after therapy (a–h), and granulocytes of a non-neutropenic mother of a dygranulopoietic neutropenia pa‐ tient with dysgranulopoiesis (i). Normal cell cycle of granulocytic cells (G0 phase) (a). Granulocytes of patient YP with chronic ITP while taking oral steroids on different occasions (b, c). Mononuclear cells (d) tested on the same occasion with (c). Granulocytes of patient AT with chronic ITP. Tested 10 days after he received anti-D therapy (e). Granulo‐ cytes of IY with acute ITP, before mega dose methyl prednisolone (MDMP) therapy (f, g); 1 week after MDMP therapy ended (h). Granulocytes of non-neutropenic mother of a congenital dysgranulopoietic neutropenia patient who had myelodysplasia (i).

The most striking changes in the bone marrow was in the erythropoietic lineage as erythroid hyperplasia, ineffective erythropoiesis and dysmorphism in erythroid lineage including ring sideroblasts (positive by 75%) and dysmorphic granulopoietic cell lineages. There was no morphologic abnormality in megakaryocytes [50], but the megakaryocytes reportedly increased or markedly decreased [27]. Megaloblastic changes were associated [103] and not associated [50] with folic acid and/or vitamin B12 levels.

Serum iron levels which were elevated in the majority (being the most prominent in those with fatty liver and typical cirrhosis), iron granules in plasma cells [50] macrophages and endothe‐ lial cells [27], large numbers of sideroblasts along with ring sideroblasts [50] were other characteristics of alcohol effect. In Zieve's syndrome, excess iron-laden foamy macrophages were encountered [27]. No correlation was found between serum iron and ring sideroblasts in the bone marrow [50].

The normal colonies of all hemopoietic cell lines and cell culture ratios in alcohol-dependent individuals showed that alcohol exerted its toxic effect not on committed stem cells but peripheral cells also [50]. Reversible bone marrow aplasia due to alcohol was also reported [27].

Cytoplasmic vacuolization was reported to be due to inhibition of acetaldehyde dehydrogen‐ ase and thus reduced rate of degradation of acetaldehyde.

Alcohol has toxic effect on cell division giving rise to arrest in cell division and multinucleated erythroblasts; direct antifolate effect on nucleic acid metabolism leading to development of megaloblasts. Additionally, it impairs iron utilization giving rise to large number of sidero‐ blasts and ring sideroblasts through disrupting pyridoxine kinase inhibiting delta aminole‐ vulinic acid synthetase which is necessary for heme synthesis [50].

Alcohol-induced hematopoietic abnormalities closely mimick those of MDS-RARS. That alcohol-induced bone marrow damage is always reversible if the patient stops to drink alcohol, and that cell culture of alcohol dependent people show normal colonies of all hematopoietic cell lines are two important points for differentiation between alcohol-induced cell damage and MDS-RARS [50].

#### *2.9.4. Smoking*

*2.9.3. Alcohol*

68 Myelodysplastic Syndromes

myelodysplasia (i).

[27].

Anemia with/without other cytopenias or pancytopenia were reported in alcohol dependent patients. Anemia was normochromic or macrocytic with round macrocytes (unlike oval macrocytes of megaloblastic anemia) [27, 50]. Vacuolated neutrophils, stomatocytes, sometimes target cells were evident. In hemolytic anemia and hyperlipidemia due to alcoholic liver disease, spherocytes, irregularly contracted cells (Zieve's syndrome) were demonstrated

**Figure 10.** Various cell cycle configurations in patients with myelodysplasia (personal archives). Cell cycle analysis of granulocytic and mononuclear cells of children with acute or chronic immune thrombocytopenic purpura (ITP) before, during and after therapy (a–h), and granulocytes of a non-neutropenic mother of a dygranulopoietic neutropenia pa‐ tient with dysgranulopoiesis (i). Normal cell cycle of granulocytic cells (G0 phase) (a). Granulocytes of patient YP with chronic ITP while taking oral steroids on different occasions (b, c). Mononuclear cells (d) tested on the same occasion with (c). Granulocytes of patient AT with chronic ITP. Tested 10 days after he received anti-D therapy (e). Granulo‐ cytes of IY with acute ITP, before mega dose methyl prednisolone (MDMP) therapy (f, g); 1 week after MDMP therapy ended (h). Granulocytes of non-neutropenic mother of a congenital dysgranulopoietic neutropenia patient who had

The most striking changes in the bone marrow was in the erythropoietic lineage as erythroid hyperplasia, ineffective erythropoiesis and dysmorphism in erythroid lineage including ring sideroblasts (positive by 75%) and dysmorphic granulopoietic cell lineages. There was no morphologic abnormality in megakaryocytes [50], but the megakaryocytes reportedly increased or markedly decreased [27]. Megaloblastic changes were associated [103] and not

Serum iron levels which were elevated in the majority (being the most prominent in those with fatty liver and typical cirrhosis), iron granules in plasma cells [50] macrophages and endothe‐ lial cells [27], large numbers of sideroblasts along with ring sideroblasts [50] were other

associated [50] with folic acid and/or vitamin B12 levels.

Moderate leukocytosis being mainly due to neutrophilia and lymphocytosis was reported. Bone marrow biopsies of 32 smokers showed normal or slightly increased cellularity, modest and mild increase in granulopoiesis and erythropoiesis respectively, right shift of granulo‐ poietic cells (the half being mature segmented neutrophils).

The special appearing macrophages with intracytoplasmic small, polygonal corpuscles showing neutrophils were striking. It was postulated that smoking inhibited locomotion of the segmented neutrophils leading to granulocytopoietic hyperplasia and accumulation of mature neutrophils in the bone marrow. These neutrophils were broken-down when they got senescent and were phagocytosed by these macrophages, resulting in "smokers' dysmyelo‐ poiesis" [51].

On the other hand, both smoking and alcohol intake were shown to constitute risk factors for MDS [104].

#### *2.9.5. Arsenic*

Arsenic can cause mono-, bi-, and pancytopenia with marked dyserythropoiesis or megalo‐ blastosis. Pancytopenia with trilineage dysplasia in the bone marrow mimicking MDS was reported. Associated symptoms like long-lasting gastrointestinal and neurological symptoms, arsenic in the urine analysis, favorable response to British anti-Lewisite (BAL) help the clinician distinguish between the two entities, although neurological symptoms may progress [27, 52].

#### *2.9.6. Lead*

Lead poisoning leads to sideroblastic anemia, microcytosis in addition to basophilic stippling and hemolytic anemia [27]. It can mimick MDS.

#### *2.9.7. Other drugs*

Isoniazid causes sideroblastic anemia. Antibiotiotic linezolid can give rise to vacuolization in elytroid precursors and ring sideroblasts together with anemia or pancytopenia. Chloram‐ phenicol makes mild bone marrow suppression with ring sideroblasts and vacuolation in erythroid and granulocyte precursors. Sodium stibogluconate, prescribed in leishmaniasis causes erythroblast karyorrhexis and severe anemia [27]. Nitrous oxide (laughing gas) which has an anesthetic and recreational use and inactivator of hydroxycobalamin may give rise to megaloblastic anemia and vitamin B12-deficiency related neurological and hematological effects associated with heavy use by healthcare workers who inhalate it in operating rooms or intensive care units [27, 53].

Granulocyte colony stimulating factor (G-CSF) and GM-CSF cause neutrophil vacuolation and dysplastic neutrophils with abnormal lobulation and development of macropolycytes, in addition to neutrophilia, eosinophilia, toxic granulation and blasts in the peripheral blood, the latter mimicking progression of leukemia or MDS [54]. Antiepileptic drugs also can give rise to cytopenia and multilineage dysplasia (personal experience).

#### **2.10. Posttransplantation**

Dysplastic findings were demonstrated after solid organ transplantation, like liver, kidney, heart, and lung. Clatch et al. [55] reported bone marrow findings of 17 liver transplantation patients taken before or 1–1288 days after orthotopic liver transplantation (post-OLT) when they developed mono-, pancytopenia or fever. The patients had received cyclosporine A and prednisolone, numerous antibiotics intermittently and a few additionally received Muromo‐ nab-CD3 and/or antilymphocyte globulin.

While dysplastic hematopoiesis was completely absent from biopsies of patients with endstage liver disease obtained before transplantation, significant trilineage dysplasia was a consistent finding in all patients who underwent OLT. Megaloblastic erythropoiesis, the most characteristic finding, macrocytosis, dysynchronous nuclear-to-cytoplasmic maturation and significant nuclear budding or bilobation were striking. Typical megaloblastic changes of the myeloid series were absent. Dysynchronous myeloid maturation, as a left shift giving rise to decreased bands and mature neutrophils, additionally dysmyelopoiesis and dysmegakaryo‐ poiesis were evident [55].

The authors postulated that iatrogenically-induced T-cell dysfunction in transplanted patients which gave rise to alterations in microenvironment, direct pharmacological toxicities and the effects of secondary infections on hematopoietic cells might have had roles in the etiology [55]. After bone marrow or hematopoietic stem cell transplantation, bone marrow was severely hypoplastic and after successful engraftment was achieved, all hematopoietic cells, mostly the erythropoietic cells appeared dysmorphic. During the following months, striking and transient increase in hematogones, mimicking leukemia was observed. During engraftment, bone marrow architecture showed various alterations, some of which were more striking in leukemia patients whose stromal cells had been damaged during previous chemotherapies [27].

Secondary MDS may develop after autologous stem cell transplantation, due to previously damaged stem cells by chemotherapy. Cytogenetic and molecular studies are useful for distinction between secondary MDS and usual secondary myelodysplasia of early posttrans‐ plantation [27].

#### **2.11. Other disorders with secondary dysplastic features**

*2.9.6. Lead*

*2.9.7. Other drugs*

70 Myelodysplastic Syndromes

intensive care units [27, 53].

**2.10. Posttransplantation**

poiesis were evident [55].

nab-CD3 and/or antilymphocyte globulin.

and hemolytic anemia [27]. It can mimick MDS.

Lead poisoning leads to sideroblastic anemia, microcytosis in addition to basophilic stippling

Isoniazid causes sideroblastic anemia. Antibiotiotic linezolid can give rise to vacuolization in elytroid precursors and ring sideroblasts together with anemia or pancytopenia. Chloram‐ phenicol makes mild bone marrow suppression with ring sideroblasts and vacuolation in erythroid and granulocyte precursors. Sodium stibogluconate, prescribed in leishmaniasis causes erythroblast karyorrhexis and severe anemia [27]. Nitrous oxide (laughing gas) which has an anesthetic and recreational use and inactivator of hydroxycobalamin may give rise to megaloblastic anemia and vitamin B12-deficiency related neurological and hematological effects associated with heavy use by healthcare workers who inhalate it in operating rooms or

Granulocyte colony stimulating factor (G-CSF) and GM-CSF cause neutrophil vacuolation and dysplastic neutrophils with abnormal lobulation and development of macropolycytes, in addition to neutrophilia, eosinophilia, toxic granulation and blasts in the peripheral blood, the latter mimicking progression of leukemia or MDS [54]. Antiepileptic drugs also can give rise

Dysplastic findings were demonstrated after solid organ transplantation, like liver, kidney, heart, and lung. Clatch et al. [55] reported bone marrow findings of 17 liver transplantation patients taken before or 1–1288 days after orthotopic liver transplantation (post-OLT) when they developed mono-, pancytopenia or fever. The patients had received cyclosporine A and prednisolone, numerous antibiotics intermittently and a few additionally received Muromo‐

While dysplastic hematopoiesis was completely absent from biopsies of patients with endstage liver disease obtained before transplantation, significant trilineage dysplasia was a consistent finding in all patients who underwent OLT. Megaloblastic erythropoiesis, the most characteristic finding, macrocytosis, dysynchronous nuclear-to-cytoplasmic maturation and significant nuclear budding or bilobation were striking. Typical megaloblastic changes of the myeloid series were absent. Dysynchronous myeloid maturation, as a left shift giving rise to decreased bands and mature neutrophils, additionally dysmyelopoiesis and dysmegakaryo‐

The authors postulated that iatrogenically-induced T-cell dysfunction in transplanted patients which gave rise to alterations in microenvironment, direct pharmacological toxicities and the effects of secondary infections on hematopoietic cells might have had roles in the etiology [55].

to cytopenia and multilineage dysplasia (personal experience).

Multiorgan failure, autoimmune lymphoproliferative syndrome associated with Fas or Fas ligand deficiency can give rise to secondary myelodysplasia. Hypothermia can lead to sideroblastic anemia with recurrent thrombocytopenia [27].

#### **3. Cases with hypoplastic bone marrow mimicking hypocellular MDS**

#### **3.1. Hypoplastic MDS and severe aplastic anemia (SAA)**

In childhood MDS, RA (RCC) subgroup constitutes the majority of patients and bone marrow cellularity was reported decreased in nearly 50% [9] and 81% [4] of children with low-grade MDS. On the other hand, hypocellular MDS (H-MDS) in adulthood is encountered in the elderly.

Differentiation between H-MDS/hypocellular RCC and AA may be challenging both in adults and children.

There are many nonhematological factors that can give rise to bone marrow hypoplasia in childhood like any type of infections (vitamin deficiencies, metabolic disorders like mevalo‐ nate kinase deficiency, rheumatic disease, mitochondrial deletions (Pearson syndrome)). Moreover, there are many hereditary or nonhereditary hematological disorders that should be differentiated from RCC in the setting of hypoplastic bone marrow like inherited bone marrow failure syndromes [4].

The most recent histopathologic criteria to distinguish RCC from SAA in childhood is presented in **Table 7** [14, 57].

In adulthood, a similar study described standardized criteria to distinguish hypocellular AML from H-MDS and aplastic anemia (AA) (**Table 8**) [59].


**Table 7.** Histopathologic criteria for differential diagnosis of SAA and RCC [4, 14, 57, 105].


**Table 8.** Recommendation for standardized approach to distinguish hypocellular AML from hypocellular MDS and AA [59].

In addition to the parameters listed in **Tables 7** and **8**, presence of ALIP, abnormal localization of megakaryocytes, erythroblast clusters, fibrosis, abnormal karyotype were also reported as parameters to be used to distinguish H-MDS and SAA (**Table 8**) [59]. Nevertheless, further studies are needed to examine the validity of histopathologic approach to hypocellular RCC and AA [60].

#### **3.2. Inherited bone marrow failure (IBMF) disorders in the differential diagnosis of hypocellular RCC**

**Refractory cytopenia of childhood (RCC) Severe aplastic anemia (SAA)**

1. Erythroid lineage

2. Granulocytic lineage -Missing or marked decrease

3. Megakaryocyte lineage -Missing or very few -No dysplastic changes -No micromegakaryocytes

1. Lymphocyte lineage

2. CD34+ cells -No increase




Inconsistent with AA

Excludes AA

MDS or AML




Dysplasia of either granulocytes or megakaryocytes in the bone marrow (if erythroid hyperplasia is the sole finding, dysplasia in erythroid lineage

Presence of any abnormal sideroblasts (>5 granules around the nucleus

Presence of two or more clusters of immature precursors (being minimum three blast per

**Table 7.** Histopathologic criteria for differential diagnosis of SAA and RCC [4, 14, 57, 105].

**Manifestation Indicative of** Presence of unequivocal blasts in the peripheral blood MDS or AML Hypogranular neutrophils or pseudo-Pelger neutrophils (>10%) MDS or AML

1–2 cm core biopsy demonstrating four to five undistorted fields (×100 magnification) Reliability in diagnosis

Consensus diagnosis required by at least 5/7 participants Reliability in diagnosis

In addition to the parameters listed in **Tables 7** and **8**, presence of ALIP, abnormal localization of megakaryocytes, erythroblast clusters, fibrosis, abnormal karyotype were also reported as

**Table 8.** Recommendation for standardized approach to distinguish hypocellular AML from hypocellular MDS and

Presence of >1–20% blasts in the bone marrow + dysplasia MDS

Differences 1. Erythroid lineage

72 Myelodysplastic Syndromes

Similarities 1. Lymphoid lineage

should be moderate to severe)

cluster) in bone marrow biopsy

AA [59].

or constituted at least 1/3 of the circumference

2. CD34+ cells -No increase

erythroid precursors) -Maturation arrest -Increased mitosis

2. Granulocytic lineage -Marked decrease -Left shift

3. Megakaryocytic lineage -Marked decrease -Micromegakaryocytes -Dysplastic findings

Several children who are diagnosed with hypoplastic RCC, may actually have one of inherited bone marrow failure (IBMF) disorders which have not been diagnosed yet. Hence, 15% of patients diagnosed with hypoplastic RCC and 2, 5, and 10% of patients diagnosed with hypoplastic RCC or aplastic anemia were later diagnosed as Fanconi anemia and heterozygous or homozygous dyskeratosis congenita [4, 9]. Inherited bone marrow failure syndromes with pancytopenia like Fanconi anemia, dyskeratosis congenita, Shwachman Diamond syndrome, amegakaryocytic thrombocytopenia (in progression), and pancytopenia with radio-ulnar synostosis display common manifestations with hypoplastic RCC, such as macrocytosis, elevated HbF, common bone marrow features. Therefore, a careful family and past history, physical examination is essential. Laboratory and molecular studies like chromosome break‐ age test and telomere length assay should be carried on, since not all children with IBMF syndromes have phenotypic characteristics [4, 9]. These diseases can progress to MDS gaining chromosomal abnormalities specific to MDS and 3q26 segment in Fanconi anemia. However, abnormal clones can also regress in any IBMF syndrome [4].

In childhood, in the setting of hypocellular bone marrow with absence of cytogenetic abnor‐ mality [4] or a bone marrow biopsy with topography and cellularity of the local hematopoiesis [9], two bone marrow biopsies at least two weeks apart are necessary [4].

### **4. Transient chromosome abnormalities in the setting of cytopenia/ spontaneous remission in MDS**

#### **4.1. Transient MDS with/without chromosomal alterations**

Monosomy 7 is an harbinger of poor prognosis and higher risk of transformation to high-risk MDS and AML [4, 61–67, 69, 70] than other chromosomal abnormalities and normal karyotype [4, 105]. The estimated time of progression in children with monosomy 7 was reported as 1.9 years, with a cumulative progression incidence of 80% at the sixth year of diagnosis [4].

In the literature, we found 13 patients who presented with MDS (n:12) or MDS-like features (n:1) and had genetic abnormalities but achieved remission only after symptomatic (n:12) and vitamin B12 and folic acid (n:1) therapies. The patients had abnormalities of chromosome 7 (12 out of 13, as −7, −7q, i7) and 11q23 translocation (1 out of 13), and +21 (1 out of 13, coexistent with −7). Two additional MDS patients (RA, RAEB) with normal karyotype achieved sponta‐ neous remission (**Table 9**).

Spontaneous disappearance of abnormal clones were reported previously in AML, EBVassociated myeloproliferative disorder and Fanconi anemia [65]. Development of cytogenetic abnormality is only one step (first hit) in the progression of malignant clone. In order to gain growth advantage, the transforming cells need other molecular changes within the cells and in the marrow microenvironment (second hit). The cases who attained spontaneous remission suggest that the development of cytogenetic abnormality might have not been supported by the other cellular and microenvironmental changes [65, 66]. Additionally, the mutation may have developed in a hematopoietic cell with limited self-renewal capacity and may not have involved the whole stem cell pool [66]. Two cases of Bader-Meunier et al. [68] suggest that patients with MDS who do not show any chromosomal abnormality can also achieve complete remission (**Table 9**). These cases show that patients who are stable should be closely observed for some time before potentially toxic therapies are started.



† The subgroup has not been reported. Probably corresponds to RCC subgroup in childhood and RCMD or MDS-U subgroups in adulthood MDS, according to the WHO 2008 classification.

©Cytogenetic abnormality found sustained on 13th month after hematologic recovery.

\* End-stage renal failure.

\*\*The patient received long-term immunosuppressive therapy with cyclosporine (for 19 months), later azacytidine (Aza) (for 6 years).
